Transgenic non-human animals for pharmacological and toxicological studies

ABSTRACT

The present invention is directed to the production, breeding and use of transgenic non-human animals such as mice in which specific genes or portions of genes have been replaced by homologues from another animal to make the physiology of the animals so modified more like that of the other animal with respect to drug pharmacokinetics and metabolism. The invention also extends to the use of the genetically modified non-human animals of the invention for pharmacological and/or toxicological studies.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/475,069,filed Apr. 30, 2004, which is United States National Phase under 35U.S.C. §371 of International Application PCT/AU02/00485, filed Apr. 18,2002 designating the U.S., and published in English as WO 02/083897 onOct. 24, 2002, which claims priority to Australian Patent ApplicationNo. PR4467, filed Apr. 18, 2001.

FIELD OF THE INVENTION

This invention relates generally to non-human animals into which foreignnucleic acid has been introduced to produce transgenic animals. Morespecifically, the invention relates to the production, breeding and useof transgenic non-human animals such as mice in which specific genes orportions of genes have been replaced by homologues from another animalto make the physiology of the animals so modified more like that of theother animal with respect to drug pharmacokinetics and metabolism. Theinvention also extends to the use of the genetically modified non-humananimals of the invention for pharmacological and/or toxicologicalstudies

DESCRIPTION OF THE RELATED ART

The cost of bringing a new drug to the market is extremely high.Typically, a pharmaceutical company will screen hundreds to hundreds ofthousands of compounds, in order to choose a single drug for marketing.Initial screening is performed in vitro with the most promisingcompounds progressing to animal studies. It is on the basis of theseanimal studies that the best drug(s) is chosen for further developmentand clinical trials. Since a considerable amount of the cost associatedwith drug development occurs subsequent to the animal studies, theaccuracy of the animal model at predicting a drug's behaviour in humans,is of obvious importance.

During drug discovery and development, animal models are used in aniterative process of characterising the drug candidates. Initial animalstudies determine the pharmacokinetics (the kinetics of drug absorption,distribution throughout the body and its eventual elimination from thebody). Subsequent animal studies measure pharmacodynamics (mechanisms ofdrug action, and the relationship between drug concentration andeffect). Typically, these studies also look at efficacy (e.g. does thecompound block tumour growth, or is the compound effective in combatingneurological disorders), short-term toxicity, optimal dosing andscheduling etc. Based on these animal studies, the most promisingcompound(s) is further developed. This stage involves continued animalstudies (e.g. longer term toxicity studies, exhaustive metabolicstudies, multiple-administration pharmacokinetic studies, expandedefficacy studies often including drug combination studies), chemicaldevelopment (e.g. production) and pharmaceutical development (e.g. drugformulation and delivery). Compounds that successfully complete all ofthese stages are then tested in human patients (Phase I-III clinicaltrials). A successful Phase I trial would demonstrate good tolerability,suitable pharmacokinetics, and in some cases demonstrate the intendedpharmacodynamic properties in humans. Such a drug would progress toPhase II and Phase III clinical trials for testing of the optimal dosingregime as well as efficacy in the treatment of disease.

The development of a single drug often requires the testing of manydifferent compounds in mice or other animal species. These animalstudies determine the choice of compound for further development andclinical trials. The main reasons for drug failure at the clinical trialstage are inappropriate pharmacokinetics and toxicological effects(often both are related to drug metabolism), and to a lesser extent,lack of efficacy due to failed concept or lack of pharmacodynamiceffect. That compounds progress to clinical trials and then fail at thislate stage is usually due to the poor predictability of existing animalmodels.

An incorrect decision, based on data from an animal model that did notaccurately reflect drug behaviour in humans, can waste vital resources,many millions of dollars and years of labour. Moreover, theopportunities lost by not pursuing other candidate compounds couldpotentially cost billions in lost revenue. For example, current mousemodels often (and unpredictably) do not accurately reflect human drugpharmacokinetics, metabolism and toxicology. Many drugs show promisingresults in mice but fail to work effectively in humans. Similarly, otherdrugs, which failed in mice and consequently were rejected, may haveworked well in humans. Thus, the process of selecting candidate drugsfor further development and clinical trials is currently based on dataobtained from a flawed animal model. The consequences of this include;a) wastage of valuable resources pursuing drugs that will not work inhumans; b) lost opportunities by not pursuing drugs which would work inhumans and c) exposure of patients to unknown risks in Phase 1 clinicaltrials.

An improved animal model, which more accurately predicts the behaviour(e.g. distribution, metabolism, efficacy and toxicity) of drugs in theanimals of interest (e.g. humans and other mammals including livestockanimals and companion animals) would provide enormous benefit to boththe pharmaceutical and/or veterinary industries and to the treatment ofdiseases affecting those animals.

SUMMARY OF THE INVENTION

Accordingly, in one aspect of the present invention, there is providedthe use of a transgenic non-primate mammal for predicting the likelybehaviour of a drug in a selected species of primate, as part of a drugscreening or evaluation process, the transgenic mammal expressing atleast a portion of a foreign polypeptide that is associated with drugbehaviour and/or metabolism, and that is expressed naturally in theselected species of primate or in a primate of a different species, orthat otherwise corresponds to the naturally expressed polypeptide,wherein the expression of an endogenous homologue of the foreignpolypeptide in the transgenic mammal is abrogated or otherwise reduced,and wherein the foreign polypeptide is other than the intended target ofthe drug. The foreign polypeptide is suitably selected from adrug-binding polypeptide, a drug-metabolising polypeptide, adrug-binding and drug-metabolising polypeptide or a drug-transportingpolypeptide.

Suitably, the transgenic mammal lacks the ability to produce afunctional endogenous polypeptide or detectable levels of the endogenouspolypeptide. The foreign polypeptide is preferably a functionalhomologue of the endogenous polypeptide. In one embodiment of this type,the foreign polypeptide is an orthologue of the endogenous polypeptide.In another embodiment of this type, the foreign polypeptide is aparalogue of the endogenous polypeptide.

The transgenic mammal may comprise an alteration to its genome, whereinthe alteration comprises replacement of the endogenous gene encoding theendogenous polypeptide with a transgene comprising a nucleotide sequenceencoding the foreign polypeptide. Alternatively, the alteration maycomprise a disruption in said endogenous gene. Suitably, the disruptionresults in reduced expression levels of the endogenous polypeptide. In apreferred embodiment, the disruption results in abrogated expressionlevels of the endogenous polypeptide. In another preferred embodiment,the mammal lacks the ability to produce a functional endogenouspolypeptide. In another embodiment, the disruption comprises a deletionof at least a portion of the endogenous gene. Suitably said deletioncomprises a deletion of nucleotide sequences encoding a region or domainof the endogenous polypeptide. Alternatively, the deletion comprises adeletion of a regulatory polynucleotide that controls at least in partthe expression of the endogenous gene. In a preferred embodiment, thedeletion comprises a deletion of the entire open reading frame encodingthe endogenous polypeptide.

Suitably, the nucleotide sequence, encoding the foreign polypeptide, isoperably linked to a regulatory polynucleotide. The regulatorypolynucleotide may comprise a nucleotide sequence of 1-10 kb. Theregulatory polynucleotide is preferably a polynucleotide that isnaturally present in the transgenic mammal or in the selected species ofprimate. In one embodiment, the regulatory polynucleotide is anendogenous polynucleotide of the transgenic mammal, or ancestor thereof.In another embodiment, the regulatory polynucleotide is an endogenouspolynucleotide of the selected species of primate. In a preferredembodiment, the regulatory polynucleotide comprises a nucleotidesequence that is naturally located upstream of the coding sequence ofthe endogenous gene. In another preferred embodiment, the regulatorypolynucleotide comprises a nucleotide sequence that is naturally locatedupstream of the coding sequence of a gene encoding the foreignpolypeptide. In an alternative embodiment, the regulatory polynucleotideis derived from an animal or source other than an animal selected fromsaid transgenic mammal, an ancestor of the transgenic mammal or theselected species of primate. In another embodiment, the regulatorypolynucleotide comprises an inducible promoter (e.g. metallothioneinpromoter).

Suitably, the alteration has been introduced into the genome of thetransgenic mammal by homologous recombination, random integration or theuse of a recombinase system (e.g. Cre-loxP or FLP-FRT system) with anucleic acid construct, comprising the transgene, in an embryonic stemcell such that the construct is stably integrated in the genome of themammal.

The transgenic animal may be heterozygous, but is preferably homozygous,for the transgene.

In one embodiment, the transgenic animal is selected from the orderRodentia. In a particularly preferred embodiment, the transgenic animalis a mouse.

In a preferred embodiment, the selected species of primate is human.

The foreign polypeptide may be selected from a serum albumin, an□-acidic glycoprotein (AGP), a cytochrome p450 (CYP), a uridinediphosphoglucuronosyl transferase (UGT), a multidrug-resistance (MDR)protein including multidrug-resistance-associated proteins (MRPs), anacetyl-transferase, a prenyl protein transferase, a peptidase, anesterase, an acetylase, a glucuronidase, a glutathione S-transferase, ora polypeptide that facilitates or catalyses a reaction selected from anoxidative reaction, a conjugation reaction, a hydrolytic reaction, areductive metabolism or other catabolic or anabolic reaction involving axenobiotic. In one embodiment, the foreign polypeptide is serum albumin,which is preferably but not exclusively human serum albumin. The humanserum albumin preferably comprises the sequence set forth in SEQ ID NO:2. In a preferred embodiment, the nucleotide sequence encoding the humanserum albumin comprises the sequence set forth in any one of SEQ ID NO:1 and 3. In another preferred embodiment, the expression of endogenousserum albumin is altered. Suitably, the endogenous serum albumin is amouse serum albumin comprising the sequence set forth in SEQ ID NO: 6.Preferably, the endogenous gene for mouse serum albumin encodes atranscript comprising the sequence set forth in SEQ ID NO: 5. Theregulatory polynucleotide suitably comprises a nucleotide sequence thatis naturally located upstream of the coding sequence relating to theendogenous gene. Preferably, the regulatory polynucleotide comprises thesequence as set forth in SEQ ID NO: 7.

In another embodiment, the foreign polypeptide is an alpha acidicglycoprotein (AGP). In a preferred embodiment of this type, the foreignpolypeptide is a human AGP selected from AGP-1, AGP-2 and AGP-3.Suitably, the human AGP-1 (also known as orosomucoid (ORM)-1) comprisesthe sequence set forth in SEQ ID NO: 14. Preferably, the nucleotidesequence encoding the human AGP-1 comprises the sequence set forth inSEQ ID NO: 13. Suitably, the human AGP-2 (also known as ORM-2) comprisesthe sequence set forth in SEQ ID NO: 16. Preferably, the nucleotidesequence encoding the human AGP-2 comprises the sequence set forth inSEQ ID NO: 15. In another preferred embodiment, the expression of anendogenous AGP is altered. Suitably, the endogenous AGP is a mouseselected from AGP-1, AGP-2 and AGP-3. Suitably, the mouse AGP-1comprises the sequence set forth in SEQ ID NO: 10. Preferably, theendogenous gene encoding the mouse AGP-1 comprises the sequence setforth in SEQ ID NO: 9. Suitably, the mouse AGP-3 comprises the sequenceset forth in SEQ ID NO: 12. Preferably, the endogenous gene encoding themouse AGP-3 comprises the sequence set forth in SEQ ID NO: 11. Theregulatory polynucleotide, in this instance, suitably comprises anucleotide sequence that is naturally located upstream of the codingsequence relating to the gene encoding the foreign polypeptide.Preferably, the regulatory polynucleotide comprises the sequence setforth in SEQ ID NO: 21 and/or 22, which correspond to regulatorypolynucleotides located naturally upstream of the human AGP-1 and AGP-2genes, respectively.

In another aspect, the invention contemplates a transgenic non-primatemammal, or progeny thereof, for predicting the likely behaviour of adrug in a selected species of primate, the transgenic animal expressingat least a portion of a foreign drug-binding polypeptide that isexpressed naturally in the selected species of primate or in a primateof a different species, or that otherwise corresponds to the naturallyexpressed polypeptide, wherein the expression of an endogenous homologueof the foreign polypeptide in the transgenic mammal is abrogated orotherwise reduced.

In yet another aspect, the invention encompasses a transgenicnon-primate mammal, or progeny thereof, for predicting the likelybehaviour of a drug in a selected species of primate, the transgenicanimal expressing at least a portion of a foreign drug-bindingpolypeptide selected from the group consisting of a serum albumin and analpha acidic glycoprotein, wherein the drug-binding polypeptide isexpressed naturally in the selected species of primate or in a primateof a different species, or that otherwise corresponds to the naturallyexpressed polypeptide, wherein the expression of an endogenous homologueof the foreign polypeptide in the transgenic mammal is abrogated orotherwise reduced.

Preferably, the transgenic mammal further expresses at least a portionof at least one other foreign polypeptide that is associated with drugbehaviour and/or metabolism and that is expressed naturally in theselected species of primate or in a primate of a different species, orthat otherwise corresponds to the naturally expressed polypeptide,wherein the expression of a respective endogenous homologue of thecorresponding other foreign polypeptide in the transgenic mammal isabrogated or otherwise reduced. Suitably, the or each other foreignpolypeptide is selected from a drug-binding polypeptide, adrug-metabolising polypeptide, a drug-binding and a drug-metabolisingpolypeptide or a drug-transporting polypeptide. In a preferredembodiment of this type, the or each other foreign polypeptide isselected from the group consisting of a serum albumin, an alpha acidicglycoprotein, a cytochrome p450 (CYP), which a preferably selected fromselected from subfamily 3A, a uridine diphospho-glucuronosyl transferase(UGT) selected from subfamily 1A, a uridine diphospho-glucuronosyltransferase, and a multidrug-resistance protein (MDR), includingP-glycoprotein and multidrug-resistance-associated proteins (MRPs).

In yet another aspect, the invention encompasses a nucleic acidconstruct, which is preferably but not exclusively a targetingconstruct, for use in producing a transgenic non-primate mammal forpredicting the likely behaviour of a drug in a selected species ofprimate, the construct including a transgene comprising a nucleotidesequence encoding at least a portion of a foreign polypeptide that isassociated with drug behaviour and/or metabolism, and that is expressednaturally in the selected species of primate or in a primate of adifferent species or that otherwise corresponds to the naturallyexpressed polypeptide. In one embodiment, the nucleic acid construct isa targeting construct comprising two regions flanking the transgenewherein the regions are sufficiently homologous with portions of thegenome of the non-primate mammal to undergo homologous recombinationwith the portions. In a preferred embodiment of this type, the portionscomprise a sequence flanking, or contained by, the endogenous gene thatencodes a polypeptide of the non-primate mammal, which polypeptide is ahomologue of the foreign polypeptide. The transgene preferably comprisesa regulatory polynucleotide operably linked to the sequence encoding atleast a portion of the foreign polypeptide. Suitably, the nucleic acidconstruct comprises a selectable marker gene.

In a further aspect, the invention resides in a method of producing atransgenic non-primate mammal for predicting the likely behaviour of adrug in a selected species of primate, the method comprising:

-   -   providing a transgene comprising a nucleotide sequence encoding        at least a portion of a foreign polypeptide that is associated        with drug behaviour and/or metabolism, and that is expressed        naturally in the selected species of primate or in a primate of        a different species or that otherwise corresponds to the        naturally expressed polypeptide; and    -   introducing the transgene into the genome of a non-primate        mammal.

Preferably, the introduction of the transgene into the genome includesproducing a nucleic acid construct as broadly described above.

Suitably, the introduction of the transgene into the genome includesfunctionally disrupting the endogenous gene, which is preferablyachieved by disrupting the structure of the endogenous gene.Alternatively, the introduction of the transgene into the genome mayinclude inserting the transgene at a site other than that of saidendogenous gene. In one embodiment, the introduction of the transgeneinto the genome includes replacing the endogenous gene or portionthereof with the transgene. In a preferred embodiment, the function ofthe endogenous gene is disrupted using, for example, a suitabletargeting construct.

The method preferably further includes the step of introducing aselectable marker gene into the genome of the non-primate mammal. In apreferred embodiment of this type, the selectable marker gene isincorporated into a targeting construct, as for example, describedabove.

In yet a further aspect, the invention resides in a method of producinga transgenic non-primate mammal for predicting the likely behaviour of adrug in a selected species of primate, the method comprising:

-   -   providing a targeting construct including a transgene comprising        a nucleotide sequence encoding at least a portion of a foreign        polypeptide that is associated with drug behaviour and/or        metabolism, and that is expressed naturally in the selected        species of primate or in a primate of a different species or        that otherwise corresponds to the naturally expressed        polypeptide, and regions flanking the transgene wherein the        regions are sufficiently homologous with portions of the genome        of the non-primate mammal to undergo homologous recombination        with the portions; and    -   introducing the targeting construct into the genome of a        non-primate cell under conditions sufficient for the transgene        to homologously recombine into a region of the genome interposed        between the portions.

According to another aspect, the invention provides a method ofproducing a transgenic non-primate mammal for predicting the likelybehaviour of a drug in a selected species of primate, the methodcomprising:

-   -   providing a nucleic acid construct including a transgene        comprising a nucleotide sequence encoding at least a portion of        a foreign polypeptide that is associated with drug behaviour        and/or metabolism, and that is expressed naturally in the        selected species of primate or in a primate of a different        species or that otherwise corresponds to the naturally expressed        polypeptide; and    -   introducing the construct into the genome of a non-primate cell        under conditions such that the transgene is randomly integrated        into the genome.

In yet a further aspect, the invention resides in a method of producinga transgenic non-primate mammal for predicting the likely behaviour of adrug in a selected species of primate, the method comprising:

-   -   providing a targeting construct including a transgene comprising        a nucleotide sequence encoding at least a portion of a foreign        polypeptide that is associated with drug behaviour and/or        metabolism and that is expressed naturally in the selected        species of primate or in a primate of a different species or        that otherwise corresponds to the naturally expressed        polypeptide, and regions flanking the transgene wherein the        regions are sufficiently homologous with portions of the genome        of the non-primate mammal to undergo homologous recombination        with the portions, wherein the portions flank, or are contained        within, the endogenous gene encoding at least a portion of a        polypeptide of the non-primate mammal, which polypeptide is a        homologue of the foreign polypeptide; and    -   introducing the targeting construct into the genome of a        non-primate cell under conditions sufficient for the transgene        to homologously recombine into at least one of the alleles of        the endogenous gene in the genome of the cell to thereby produce        a cell containing at least one allele of the endogenous gene        replaced, or disrupted, with the transgene.

The present invention further resides in a method of producing atransgenic non-primate mammal for predicting the likely behaviour of adrug in a selected species of primate, the method comprising:

-   -   providing a first targeting construct including a transgene        comprising a nucleotide sequence encoding at least a portion of        a foreign polypeptide that is associated with drug behaviour        and/or metabolism and that is expressed naturally in the        selected species of primate or in a primate of a different        species or that otherwise corresponds to the naturally expressed        polypeptide, wherein the transgene is flanked by portions of the        genome of a non-primate cell; and    -   providing a second targeting construct comprising: i) at least a        portion of the endogenous gene encoding an endogenous        polypeptide that is a homologue of the foreign polypeptide;        and ii) a polynucleotide capable of disrupting the endogenous        gene;    -   introducing the first targeting construct into the non-primate        cell under conditions sufficient for the transgene to        homologously recombine into a region of the genome of the cell,        corresponding to the portions; and    -   introducing the second targeting construct into the cell under        conditions sufficient for the polynucleotide to homologously        recombine into at least one allele of the endogenous gene in the        genome of the cell to thereby produce a cell containing at least        one disrupted allele of the endogenous gene.

The present invention further extends to a method of producing atransgenic non-primate mammal for predicting the likely behaviour of adrug in a selected species of primate, the method comprising:

-   -   providing a nucleic acid construct including a transgene        comprising a nucleotide sequence encoding at least a portion of        a foreign polypeptide that is associated with drug behaviour        and/or metabolism and that is expressed naturally in the        selected species of primate or in a primate of a different        species or that otherwise corresponds to the naturally expressed        polypeptide;    -   providing a targeting construct comprising: i) at least a        portion of the endogenous gene encoding an endogenous        polypeptide that is a homologue of the foreign polypeptide;        and ii) a polynucleotide capable of disrupting the endogenous        gene;    -   introducing the nucleic acid construct into a non-primate cell        under conditions sufficient for the transgene to randomly        integrate into a region of the genome of the cell; and    -   introducing the targeting construct into the cell under        conditions sufficient for the polynucleotide to homologously        recombine into at least one allele of the endogenous gene in the        genome of the cell to thereby produce a cell containing at least        one disrupted allele of the endogenous gene.

The cell employed in the above production method is preferably anembryonic stem cell, preferably an embryonic stem cell from a mammalwithin the order Rodentia and most preferably a mouse embryonic stemcell.

In a preferred embodiment, the method further comprises injecting theembryonic stem cell containing at least one transgene into theblastocyst or other early developmental stage of a non-human animal.

In another preferred embodiment, the method further comprisesintroducing the injected blastocyst into a pseudo-pregnant non-humananimal and permitting the pseudo-pregnant animal to deliver progenycontaining at least one homologously recombined transgene.

In yet another preferred embodiment, the progeny containing the at leastone homologously recombined transgene is further characterised byexpressing at least a portion of the foreign polypeptide at detectablelevels.

In another preferred embodiment, the progeny containing the at least onehomologously recombined transgene is further characterised by expressingreduced or undetectable levels of the endogenous polypeptide.

In an alternative preferred embodiment, the progeny lacks the ability toproduce functional endogenous polypeptide.

The method may further include the step of breeding a transgenicnon-primate mammal produced by a method as broadly described above andproducing progeny of that mammal. For example, mammals containing thesame transgene can be inbred to produce mammals that are homozygous forthe transgene. Alternatively or additionally, transgenic mammalscontaining different transgenes described in this invention can beinterbred to produce mammals containing two or more differenttransgenes. Alternatively or additionally, any of these transgenicmammals can be crossbred with any other genetically modified, wild-typeor mutant mammals of the same species in order to obtain mammalscontaining the transgene(s) described in the present invention as wellas the desired genetic characteristics of the other mammals used in thecrossbreeding strategy. When the transgenic mammal is a mouse,crossbreeding strategies may include crossbreeding the transgenic mousewith another mouse including, but not restricted to, a nude mouse, aSCID mouse, an inbred strain of mouse such as BALB/c, a mouse designedto mimic a specific human disease or a mouse with a useful reporterconstruct.

The transgenic mammals and cells derived therefrom are useful forscreening biologically active agents including drugs and forinvestigating their distribution, efficacy, metabolism and/or toxicity.These screening methods are of particular use for assessing withimproved predictability the behaviour of a drug in the primate speciesof interest. Accordingly, in yet a further aspect, the inventionfeatures a method of assessing the behaviour of a drug in a selectedspecies of primate, as part of a drug screening or evaluation process,comprising administering a drug to a transgenic non-primate mammalexpressing at least a portion of a foreign polypeptide that isassociated with drug behaviour and/or metabolism, and that is expressednaturally in the selected species of primate or in a primate of adifferent species or that otherwise corresponds to the naturallyexpressed polypeptide, and wherein the foreign polypeptide is other thanthe intended target of the drug, and conducting analytical tests todetermine the behaviour of the drug in the transgenic mammal, theresults of which have a higher correlation to the behaviour of the drugin the selected species of primate than the results obtained from amammal of the same species as the transgenic mammal, which expresses theendogenous polypeptide but which does not express the foreignpolypeptide or portion thereof.

In one embodiment, the analytical test comprises assessing directly orindirectly, the concentration and/or distribution of the drug in thetransgenic mammal to which it has been administered. In anotherembodiment, the analytical test comprises assessing directly orindirectly, the efficacy of the drug in the transgenic mammal to whichit has been

-   -   administered. In yet another embodiment, the analytical test        comprises assessing directly or indirectly, the toxicity of the        drug in the transgenic mammal to which it has been administered.        In a further embodiment, the analytical test comprises assessing        directly or indirectly, the half-life of the drug in the        transgenic mammal to which it has been    -   administered. In still a further embodiment, the analytical test        comprises assessing directly or indirectly, the pharmacodynamics        of the drug in the transgenic mammal to which it has been        administered. In still another embodiment, the analytical test        comprises assessing directly or indirectly, the pharmacokinetics        of the drug in the transgenic mammal to which it has been        administered.

The invention also encompasses the use of the transgenic mammal asbroadly described above in the study of drug behaviour.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation showing a linear map of the mouseALB gene.

FIG. 2 is a schematic representation showing a linear map of oneembodiment of a mouse ALB targeting construct for replacing the mouseALB gene with a human ALB cDNA.

FIG. 3 is a schematic representation showing a linear map of the mouseAGP locus.

FIG. 4 is a schematic representation showing a linear map of oneembodiment of a mouse AGP targeting construct for knocking out the mouseAGP locus.

BRIEF DESCRIPTION OF THE SEQUENCES SUMMARY TABLE

TABLE A SEQUENCE ID DESCRIPTION LENGTH SEQ ID NO: 1 Nucleotide sequencecorresponding to the human serum 19002 nts albumin gene as set forth inGenBank Accession No. M12523 SEQ ID NO: 2 Polypeptide encoded by SEQ IDNO: 1 609 aa SEQ ID NO: 3 Nucleotide sequence corresponding to the humanserum 2216 nts albumin mRNA as set forth in GenBank Accession No.XM_031320 SEQ ID NO: 4 Polypeptide encoded by SEQ ID NO: 3 609 aa SEQ IDNO: 5 Nucleotide sequence corresponding to mouse serum 2027 nts albumin(ALB) mRNA as set forth in GenBank Accession No. AJ011413 SEQ ID NO: 6Polypeptide encoded by SEQ ID NO: 5 608 aa SEQ ID NO: 7 Nucleotidesequence corresponding to the flanking 2079 nts sequence immediatelyupstream of the coding sequence of the mouse ALB gene, as set forth inGenBank Accession No. J04738 SEQ ID NO: 8 Nucleotide sequencecorresponding to a flanking 900 nts sequence upstream of the alphafetoprotein (AFP) gene, as set forth in GenBank Accession No. J05246 SEQID NO: 9 Nucleotide sequence corresponding to the mouse alpha- 4133 nts1-acid glycoprotein I (AGP-1) gene, as set forth in GenBank AccessionNo. M17376 SEQ ID NO: 10 Polypeptide encoded by SEQ ID NO: 9 207 aa SEQID NO: 11 Nucleotide sequence corresponding to the mouse alpha- 4002 aa1-acid glycoprotein 3 (AGP-3) gene, as set forth in GenBank AccessionNo. S38219 SEQ ID NO: 12 Polypeptide encoded by SEQ ID NO: 11 206 aa SEQID NO: 13 Nucleotide sequence corresponding to the coding 803 ntssequence of human orosomucoid 1 (ORM-1) gene, as set forth in GenBankAccession No. NM_000607 SEQ ID NO: 14 Polypeptide encoded by SEQ ID NO:13 201 aa SEQ ID NO: 15 Nucleotide sequence corresponding to the coding606 nts sequence of the human orosomucoid 2 (ORM-2) gene, as set forthin GenBank Accession No. NM_000608 SEQ ID NO: 16 Polypeptide encoded bySEQ ID NO: 15 201 aa SEQ ID NO: 17 Nucleotide sequence corresponding tothe human 4944 nts orosomucoid 2 (ORM-2) gene, as set forth in GenBankAccession No. M21540 SEQ ID NO: 18 Polypeptide encoded by SEQ ID NO: 17201 aa SEQ ID NO: 19 Nucleotide sequence corresponding to the human DNA125673 nts sequence from BAC clone RP11-82I1 relating to chromosome 9,as set forth in GenBank Accession No. AL356796 SEQ ID NO: 20 Nucleotidesequence corresponding to human AGP- 18875 nts 1-AGP-2 transgene SEQ IDNO: 21 Nucleotide sequence corresponding to human AGP-1 6032 ntspromoter SEQ ID NO: 22 Nucleotide sequence corresponding to human AGP-21944 nts promoter SEQ ID NO: 23 Nucleotide sequence corresponding tohuman DNA 123778 nts sequence from BAC clone RP11-757A13, as set forthin GenBank Accession No. AC069294 SEQ ID NO: 24 Nucleotide sequencecorresponding to human 2764 nts cytochrome P450, subfamily IIIA(niphedipine oxidase), polypeptide 4 (CYP3A4), mRNA, as set forth inGenBank Accession No. NM_017460 SEQ ID NO: 25 Polypeptide encoded by SEQID NO: 24 503 aa SEQ ID NO: 26 HALB1F primer 24 nts SEQ ID NO: 27 HALB3Rprimer 27 nts SEQ ID NO: 28 HALB5F primer 30 nts SEQ ID NO: 29 HALB4Rprimer 20 nts SEQ ID NO: 30 Forward primer corresponding to nt 1-32 ofSEQ ID 32 nts NO: 7 SEQ ID NO: 31 Reverse primer corresponding to thereverse 31 nts complement of nt 2035-2065 from SEQ ID NO: 7 SEQ ID NO:32 Forward primer corresponding to nt 1973-2002 of SEQ 32 nts ID NO: 5SEQ ID NO: 33 Reverse primer corresponding to the reverse 29 ntscomplement of nt 1-29 of SEQ ID NO: 8 SEQ ID NO: 34 Nucleotide sequencecorresponding to a sequence that 15295 nts spans the first 11 exons ofthe mouse albumin gene, as set forth in Accession No. c077802366.Contig3 SEQ ID NO: 35 Malb353F primer 23 nts SEQ ID NO: 36 Malb2382R primer 43nts SEQ ID NO: 37 Malb6310F 30 nts SEQ ID NO: 38 Malb13382R 30 nts SEQID NO: 39 Nucleotide sequence corresponding to a genomic 27781 ntssequence that spans the entire mouse albumin gene, as set forth inSanger assembly No. F105491 SEQ ID NO: 40 albt9649R primer 28 nts SEQ IDNO: 41 albt2842F primer 31 nts SEQ ID NO: 42 Nucleotide sequencecorresponding to nts 88681-130080 41400 nts of BAC279 SEQ ID NO: 43AGP99R primer 22 nts SEQ ID NO: 44 AGP45F primer 22 nts SEQ ID NO: 45AGP49R primer 22 nts SEQ ID NO: 46 AGP12F primer 19 nts SEQ ID NO: 47AGP339F primer 20 nts SEQ ID NO: 48 AGP403R primer 21 nts SEQ ID NO: 49AGP5′exF 22 nts SEQ ID NO: 50 AGP5′exR 20 nts SEQ ID NO: 51 AGP3′exF 20nts SEQ ID NO: 52 AGP3′exR 20 nts SEQ ID NO: 53 Forward primercorresponding to nt 47283-47314 of 32 nts SEQ ID NO: 19 SEQ ID NO: 54Reverse primer corresponding to the reverse 32 nts complement of nt58112-58142 of SEQ ID NO: 19 SEQ ID NO: 55 Forward primer correspondingto nt 58112-58142 of 31 nts SEQ ID NO: 19 SEQ ID NO: 56 Reverse primercorresponding to the reverse 27 nts complement of nt 66131-66157 of SEQID NO: 19 SEQ ID NO: 57 Forward primer corresponding to nt 84-111 of SEQID 28 nts NO: 9 SEQ ID NO: 58 Reverse primer corresponding to thereverse 30 nts complement of nt 2991-3020 in SEQ ID NO: 9 SEQ ID NO: 59Forward primer corresponding to nt 898-924 of SEQ ID 27 nts NO: 11 SEQID NO: 60 Reverse primer corresponding to the reverse 32 nts complementof nt 3492-3523 of SEQ ID NO: 11 SEQ ID NO: 61 Forward primercorresponding to nt 26930-26965 of 36 nts SEQ ID NO: 23 SEQ ID NO: 62Reverse primer corresponding to the reverse 28 nts complement of nt35227-35254 of SEQ ID NO: 23 SEQ ID NO: 63 Forward primer correspondingto nt 54-80 of SEQ ID 27 nts NO: 24 SEQ ID NO: 64 Reverse primercorresponding to the reverse 27 nts complement of nt 2738-2764 of SEQ IDNO: 24 SEQ ID NO: 65 Mouse UGT gene locus 75798 nts SEQ ID NO: 66ugt23275F primer 31 nts SEQ ID NO: 67 ugt27501R primer 30 nts SEQ ID NO:68 ugt35967R primer 21 nts SEQ ID NO: 69 ugt31170F primer 23 nts SEQ IDNO: 70 Human MDR-1 cDNA 4643 nts SEQ ID NO: 71 MDR1 primer 22 nts SEQ IDNO: 72 5′ flank human MDR contig 10094 nts SEQ ID NO: 73 MDR2 primer 22nts SEQ ID NO: 74 MDR3 primer 23 nts SEQ ID NO: 75 MDR4 primer 23 ntsSEQ ID NO: 76 MDR5 primer 22 nts SEQ ID NO: 77 MDR6 primer 28 nts SEQ IDNO: 78 MDR7 primer 26 nts SEQ ID NO: 79 Nucleotide sequencecorresponding to reverse 1940 nts complement of nt 36061-38001 ofAC005068 BAC clone CTB-137N13 SEQ ID NO: 80 MDR8 primer 29 nts

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“agent” means a naturally occurring or synthetically produced moleculewhich interacts either directly or indirectly with a target member, thelevel and/or functional activity of which is to be modulated.

“AGP” means the α-acidic glycoprotein family (also abbreviated as AAG).

“Antigen-binding molecule” means a molecule that has binding affinityfor a target antigen. It will be understood that this term extends toimmunoglobulins, immunoglobulin fragments and non-immunoglobulin derivedprotein frameworks that exhibit antigen-binding activity.

By “autologous” is meant something (e.g. cells, tissues etc) derivedfrom the same organism.

As used herein, the term “behaviour” when used in relation to a drugincludes but is not restricted to the distribution, half-life, efficacyand toxicity of the drug and its metabolites as well as any otherphysiological or pathological consequences of administering thedrug/compound.

As used herein, the term “cis-acting sequence” or “cis-regulatoryregion” or “regulatory region” or similar term shall be taken to meanany sequence of nucleotides, which when positioned appropriatelyrelative to an expressible genetic sequence, is capable of regulating,at least in part, the expression of the genetic sequence. Those skilledin the art will be aware that a cis-regulatory region may be capable ofactivating, silencing, enhancing, repressing or otherwise altering thelevel of expression and/or cell-type-specificity and/or developmentalspecificity of a gene sequence at the transcriptional orpost-transcriptional level.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “corresponds to” or “corresponding to” is meant a polynucleotide (a)having a nucleotide sequence that is substantially identical orcomplementary to all or a portion of a reference polynucleotide sequenceor (b) encoding an amino acid sequence identical to an amino acidsequence in a peptide or protein. This phrase also includes within itsscope a peptide or polypeptide having an amino acid sequence that issubstantially identical to a sequence of amino acids in a referencepeptide or protein.

By “derivative” is meant a polypeptide that has been derived from thebasic sequence by modification, for example by conjugation or complexingwith other chemical moieties or by post-translational modificationtechniques as would be understood in the art. The term “derivative” alsoincludes within its scope alterations that have been made to a parentsequence including additions, or deletions that provide for functionallyequivalent molecules. Accordingly, the term derivative encompassesmolecules that will modulate function and/or an immune response.

“Drug” refers to any compound, peptide, protein, lipid, carbohydrate orother molecule or moiety which alters or which is intended to alter thephysiology or pathology of an organism, organ, tissue or cell.

“Exon” means a region of DNA or the mRNA segment it encodes that ispresent in the mature mRNA molecule.

The term “foreign polynucleotide” or “exogenous polynucleotide” or“heterologous polynucleotide” refers to any nucleic acid (e.g. a genesequence) which is introduced into the genome of an animal byexperimental manipulations and may include gene sequences found in thatanimal so long as the introduced gene contains some modification (e.g. apoint mutation, the presence of a selectable marker gene, the presenceof a loxP site, etc.) relative to the naturally-occurring gene.

The term “gene” as used herein refers to any and all discrete codingregions of the cell's genome, as well as associated non-coding andregulatory regions. The gene is also intended to mean the open readingframe encoding specific polypeptides, introns, and adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation ofexpression. In this regard, the gene may further comprise endogenous(i.e. naturally associated with a given gene) or heterologous controlsignals such as promoters, enhancers, termination and/or polyadenylationsignals. The DNA sequences may be cDNA or genomic DNA or a fragmentthereof. The gene may be introduced into an appropriate vector forextrachromosomal maintenance or for integration into the host.

The term “homologue” in the context of polypeptides refers to apolypeptide of a reference animal, which has a similar sequence to theencoded amino acid sequence of a polypeptide of a different animal.Although two polypeptides are said to be “homologous”, this does notimply that there is necessarily an evolutionary relationship between theproteins. Instead, the term “homologous” is defined to mean that the twopolypeptides have similar amino acid sequences. In addition, although inmany cases polypeptides with similar amino acid sequences will havesimilar functions, the term “homologous” does not imply that thepolypeptides must be functionally similar to each other. When“homologous” is used in reference to polypeptides or peptides, it isrecognised that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g. charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (see,e.g. Pearson et al. (1994) Methods in Molecular Biology 24: 307-31). Thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. See, e.g. the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. A preferred algorithm when comparing a referencesequence to a database containing a large number of sequences fromdifferent organisms is the computer program BLAST, especially blastp ortblastn (Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402).Preferred parameters for blastp are:

Expectation value:  10 (default) Filter: seg (default) Cost to open agap:  11 (default) Cost to extend a gap:  1 (default Max. alignments:100 (default) Word size:  11 (default) No. of descriptions: 100(default) Penalty Matrix: BLOWSUM62

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Databasesearching using amino acid sequences can be measured by algorithms otherthan blastp known in the art. For instance, polypeptide sequences can becompared using Fasta, a program in GCG Version 6.1. Fasta providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson (1990) Methodsin Enzymology 183: 63-98). For example, percent sequence identitybetween amino acid sequences can be determined using Fasta with itsdefault parameters (a word size of 2 and the PAM250 scoring matrix), asprovided in GCG Version 6.1. The invention envisions two general typesof polypeptide “homologues” Type 1 homologues are strong homologues. Acomparison of two polypeptides that are Type 1 homologues would resultin a blastp score of less than 1×10⁻⁴⁰, using the blastp algorithm andthe parameters listed above. The lower the blastp score, that is, thecloser it is to zero, the better the match between the polypeptidesequences. Type 2 homologues are weaker homologues. A comparison of twopolypeptides that are Type 2 homologues would result in a blastp scoreof between 1×10⁻⁴⁰ and 1×10⁻¹⁰, using the Blast algorithm and theparameters listed above. One having ordinary skill in the art willrecognise that other algorithms can be used to determine weak or stronghomology.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or polypeptides or between two nucleic acidmolecules. Homology can be determined by comparing a position in eachsequence, which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same base or aminoacid, then the molecules are homologous at that position. A degree ofhomology between sequences is a function of the number of matching orhomologous positions shared by the sequences. An “unrelated” or“non-homologous” sequence shares less than 40% identity, thoughpreferably less than 25% identity, with a reference sequence.

“Humanised” means made more human like in function and/or structure butnot necessarily identical to the human equivalent. The term “human” asused in reference to polynucleotide or amino acid sequences, may alsoinclude any sequence that is human-like in function.

An “intron” is a region of DNA or the mRNA segment that it encodes thatis generally spliced out from the primary mRNA and is not present in themature mRNA molecule.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state.

A “knock-in” animal, as used herein, refers to a genetically modifiedanimal in which a specific gene or part thereof is replaced by a foreigngene or DNA sequence.

By “knock-out” animal is meant a genetically modified animal in which agene is removed or rendered inoperative.

The term “mammal” is used herein in its broadest sense and includesrodents, primates, ovines, bovines, ruminants, lagomorphs, porcine,caprices, equines, canines, and felines. Preferred non-human mammals areselected from the order Rodentia that includes murines (e.g. rats andmice), most preferably mice.

“Messenger RNA” or “mRNA” is the “transcript” produced in a cell usingDNA as a template, which itself encodes a protein.

The terms “metabolism”, “metabolising” and the like when used inrelation to a drug, or to a polypeptide with which it interacts, referto all aspects of biotransformation of compounds, including but notlimited to, the absorption, binding, uptake, excretion, distribution,transport, processing, conversion or degradation of exogenous agents aswell as pathological reactions resulting directly or indirectly fromadministration of the drug.

The term “5′ non-coding region” is used herein in its broadest contextto include all nucleotide sequences which are derived from the upstreamregion of an expressible gene, other than those sequences which encodeamino acid residues which comprise the polypeptide product of said gene,wherein 5′ non-coding region confers or activates or otherwisefacilitates, at least in part, expression of the gene.

“Nude mice” are a strain of immuno-incompetent mice also known asathymic mice, often used as a host for growing human tumour cells.

The term “oligonucleotide” as used herein refers to a polymer composedof a multiplicity of nucleotide units (deoxyribonucleotides orribonucleotides, or related structural variants or synthetic analoguesthereof) linked via phosphodiester bonds (or related structural variantsor synthetic analogues thereof). Thus, while the term “oligonucleotide”typically refers to a nucleotide polymer in which the nucleotides andlinkages between them are naturally occurring, it will be understoodthat the term also includes within its scope various analoguesincluding, but not restricted to, peptide nucleic acids (PNAs),phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methylribonucleic acids, and the like. The exact size of the molecule may varydepending on the particular application. An oligonucleotide is typicallyrather short in length, generally from about 10 to 30 nucleotides, butthe term can refer to molecules of any length, although the term“polynucleotide” or “nucleic acid” is typically used for largeoligonucleotides.

“Operably linked” or operably connected and the like refer to a linkageof polynucleotide elements in a functional relationship. A nucleic acidsequence is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. Operably linked meansthat the nucleic acid sequences being linked are typically contiguousand, where necessary to join two protein coding regions, contiguous andin reading frame. A coding sequence is “operably linked to” anothercoding sequence when RNA polymerase will transcribe the two codingsequences into a single mRNA, which is then translated into a singlepolypeptide having amino acids derived from both coding sequences. Thecoding sequences need not be contiguous to one another so long as theexpressed sequences are ultimately processed to produce the desiredprotein. “Operably linking” a promoter to a transcribable polynucleotideis meant placing the transcribable polynucleotide (e.g. protein encodingpolynucleotide or other transcript) under the regulatory control of apromoter, which then controls the transcription and optionallytranslation of that polynucleotide. In the construction of heterologouspromoter/structural gene combinations, it is generally preferred toposition a promoter or variant thereof at a distance from thetranscription start site of the transcribable polynucleotide, which isapproximately the same as the distance between that promoter and thegene it controls in its natural setting; i.e. the gene from which thepromoter is derived. As is known in the art, some variation in thisdistance can be accommodated without loss of function. Similarly, thepreferred positioning of a regulatory sequence element (e.g. anoperator, enhancer etc) with respect to a transcribable polynucleotideto be placed under its control is defined by the positioning of theelement in its natural setting; i.e. the genes from which it is derived.

The term “orthologue” refers to genes or proteins which are homologuesvia speciation, e.g. closely related and assumed to have common descentbased on structural and functional considerations. Orthologous proteinsfunction as recognisably the same or similar activity in differentspecies. The term “paralogue” refers to genes or proteins which arehomologues via gene duplication, e.g. duplicated variants of a genewithin a genome. See also, Fritch, W M (1970) Syst Zool 19: 99-113.

-   -   “PCR” means polymerase chain reaction, a method for amplifying        DNA.

The term “polynucleotide” or “nucleic acid” as used herein designatesmRNA, RNA, cRNA, cDNA or DNA. The term typically refers tooligonucleotides greater than 30 nucleotides in length. Polynucleotidesequences are understood to encompass complementary strands as well asalternative backbones described herein.

The terms “polynucleotide variant” and “variant” refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridise witha reference sequence under stringent conditions that are definedhereinafter. These terms also encompasses polynucleotides in which oneor more nucleotides have been added or deleted, or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletionsand substitutions can be made to a reference polynucleotide whereby thealtered polynucleotide retains the biological function or activity ofthe reference polynucleotide. The terms “polynucleotide variant” and“variant” also include naturally occurring allelic variants.

“Polypeptide”, “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues and to variants andsynthetic analogues of the same. Thus, these terms apply to amino acidpolymers in which one or more amino acid residues is a syntheticnon-naturally occurring amino acid, such as a chemical analogue of acorresponding naturally occurring amino acid, as well as tonaturally-occurring amino acid polymers.

The term “polypeptide variant” refers to a polypeptide which has somedifferences in its amino acid sequence as compared to that of areference polypeptide. Thus, a polypeptide variant is distinguished froma reference polypeptide by the addition, deletion or substitution of atleast one amino acid.

By “primer” is meant an oligonucleotide which, when paired with a strandof DNA, is capable of initiating the synthesis of a primer extensionproduct in the presence of a suitable polymerising agent. The primer ispreferably single-stranded for maximum efficiency in amplification butmay alternatively be double-stranded. A primer must be sufficiently longto prime the synthesis of extension products in the presence of thepolymerisation agent. The length of the primer depends on many factors,including application, temperature to be employed, template reactionconditions, other reagents, and source of primers. For example,depending on the complexity of the target sequence, the oligonucleotideprimer typically contains 15 to 35 or more nucleotides, although it maycontain fewer nucleotides. Primers can be large polynucleotides, such asfrom about 200 nucleotides to several kilobases or more. Primers may beselected to be “substantially complementary” to the sequence on thetemplate to which it is designed to hybridise and serve as a site forthe initiation of synthesis. By “substantially complementary”, it ismeant that the primer is sufficiently complementary to hybridise with atarget nucleotide sequence. Preferably, the primer contains nomismatches with the template to which it is designed to hybridise butthis is not essential. For example, non-complementary nucleotides may beattached to the 5′ end of the primer, with the remainder of the primersequence being complementary to the template. Alternatively,non-complementary nucleotides or a stretch of non-complementarynucleotides can be interspersed into a primer, provided that the primersequence has sufficient complementarity with the sequence of thetemplate to hybridise therewith and thereby form a template forsynthesis of the extension product of the primer.

“Probe” refers to a molecule that binds to a specific sequence orsub-sequence or other moiety of another molecule. Unless otherwiseindicated, the term “probe” typically refers to a polynucleotide probethat binds to another nucleic acid, often called the “target nucleicacid”, through complementary base pairing. Probes may bind targetnucleic acids lacking complete sequence complementarity with the probe,depending on the stringency of the hybridisation conditions. Probes canbe labelled directly or indirectly.

“Promoter” means a region of DNA, generally upstream (5′) of the mRNAencoding region, which controls the initiation and level oftranscription. Reference herein to a “promoter” is to be taken in itsbroadest context and includes the transcriptional regulatory sequencesof a classical genomic gene, including a TATA box and CCAAT boxsequences, as well as additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers) that alter geneexpression in response to developmental and/or environmental stimuli, orin a tissue-specific or cell-type-specific manner. A promoter isusually, but not necessarily, positioned upstream or 5′, of a structuralgene, the expression of which it regulates. Furthermore, the regulatoryelements comprising a promoter are usually positioned within 2 kb of thestart site of transcription of the gene. Promoters according to theinvention may contain additional specific regulatory elements, locatedmore distal to the start site to further enhance expression in a cell,and/or to alter the timing or inducibility of expression of a structuralgene to which it is operably connected.

The term “recombinant polynucleotide” as used herein refers to apolynucleotide formed in vitro by the manipulation of nucleic acid intoa form not normally found in nature. For example, the recombinantpolynucleotide may be in the form of an expression vector. Generally,such expression vectors include transcriptional and translationalregulatory nucleic acid operably linked to the nucleotide sequence.

By “recombinant polypeptide” is meant a polypeptide made usingrecombinant techniques, i.e. through the expression of a recombinantpolynucleotide.

By “reporter molecule” as used in the present specification is meant amolecule that, by its chemical nature, provides an analyticallyidentifiable signal. For example, the detection of a complex comprisingan antigen-binding molecule and its target antigen. The term “reportermolecule” also extends to use of cell agglutination or inhibition ofagglutination such as red blood cells on latex beads, and the like.

“SCID mice” means a strain of immuno-incompetent mice with SevereCombined Immuno-Deficiency.

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e. the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. For the purposes of the present invention, “sequence identity”will be understood to mean the “match percentage” calculated by theDNASIS computer program (Version 2.5 for windows; available from HitachiSoftware engineering Co., Ltd., South San Francisco, Calif., USA) usingstandard defaults as used in the reference manual accompanying thesoftware.

“Southern blot” is a method for detecting specific DNA sequences. Inbrief, a DNA sample is cut with restriction enzymes, electrophoresed,transferred to a membrane and then probed with a labelled DNA fragmentof interest.

“Standard mice” means any strain of mice not bearing the geneticmodifications of the present invention.

“Stringency” as used herein, refers to the temperature and ionicstrength conditions, and presence or absence of certain organicsolvents, during hybridisation and washing procedures. The higher thestringency, the higher will be the degree of complementarity betweenimmobilised target nucleotide sequences and the labelled probepolynucleotide sequences that remain hybridised to the target afterwashing.

“Stringent conditions” refers to temperature and ionic conditions underwhich only nucleotide sequences having a high frequency of complementarybases will hybridise. The stringency required is nucleotide sequencedependent and depends upon the various components present duringhybridisation and subsequent washes, and the time allowed for theseprocesses. Generally, in order to maximise the hybridisation rate,non-stringent hybridisation conditions are selected; about 20 to 25° C.lower than the thermal melting point (T_(m)). The T_(m) is thetemperature at which 50% of specific target sequence hybridises to aperfectly complementary probe in solution at a defined ionic strengthand pH. Generally, in order to require at least about 85% nucleotidecomplementarity of hybridised sequences, highly stringent washingconditions are selected to be about 5 to 15° C. lower than the T_(m). Inorder to require at least about 70% nucleotide complementarity ofhybridised sequences, moderately stringent washing conditions areselected to be about 15 to 30° C. lower than the T_(m). Highlypermissive (low stringency) washing conditions may be as low as 50° C.below the T_(m), allowing a high level of mismatching between hybridisedsequences. Those skilled in the art will recognise that other physicaland chemical parameters in the hybridisation and wash stages can also bealtered to affect the outcome of a detectable hybridisation signal froma specific level of homology between target and probe sequences. Otherexamples of stringency conditions are described in section 3.3.

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated. An “isolated polynucleotide”is therefore a substantially purified polynucleotide.

“t_(1/2)” means the time needed for a drug to decrease its concentrationby one-half (also known as a half-life).

“Transfection” means the process during which a nucleic acid molecule(e.g. a plasmid or DNA fragment) is inserted into a eukaryotic cell.Typically, 2-50% of cells take up the plasmid and express the proteinproduct for ˜3 days without incorporating the plasmid DNA or DNAfragment into the cell's chromosomes (=transient transfection). A smallproportion of these cells will eventually incorporate the plasmid DNAinto their chromosomes and permanently express the protein product(=stable transfection).

The term “transgene” is used herein to describe genetic material thathas been or is about to be artificially inserted into the genome of acell, particularly a mammalian cell of a living animal. The transgene isused to transform a cell, meaning that a permanent or transient geneticchange, preferably a permanent genetic change, is induced in a cellfollowing incorporation of exogenous nucleic acid (usually DNA). Apermanent genetic change is generally achieved by introduction of theDNA into the genome of the cell. Vectors for stable integration includeplasmids, retroviruses and other animal viruses, YACs (yeast artificialchromosome), BACs (bacterial artificial chromosome) and the like. Thetransgene is suitably derived from animals including, but not limitedto, vertebrates, preferably mammals such as rodents, humans, non-humanprimates, ovines, bovines, ruminants, lagomorphs, porcines, caprines,equines, canines, felines, aves, etc.

As used herein the term “transgenic” refers to a genetically modifiedanimal in which the endogenous genome is supplemented or modified by therandom or site-directed integration of a foreign gene or sequence.

The “transgenic animals” of the invention are preferably produced byexperimental manipulation of the genome of the germline of the animal.These genetically engineered animals may be produced by several methodsincluding the introduction of a “transgene” comprising nucleic acid(usually DNA) into an embryonal target cell or integration into achromosome of the somatic and/or germ line cells of a animal by way ofhuman intervention, such as by the methods described herein. Animals,which contain a transgene, are referred to as “transgenic animals”. Atransgenic animal is an animal whose genome has been altered by theintroduction of a transgene.

“UGTs” or “UDPGTs” mean uridine glucuronosyl transferases or uridinediphosphoglucuronosyl transferases.

By “vector” is meant a nucleic acid molecule, preferably a DNA moleculederived, for example, from a plasmid, bacteriophage, or plant virus,into which a nucleic acid sequence may be inserted or cloned. A vectorpreferably contains one or more unique restriction sites and may becapable of autonomous replication in a defined host cell including atarget cell or tissue or a progenitor cell or tissue thereof, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. Accordingly, the vector may be an autonomouslyreplicating vector, i.e. a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g. a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. A vector system maycomprise a single vector or plasmid, two or more vectors or plasmids,which together contain the total DNA to be introduced into the genome ofthe host cell, or a transposon. The choice of the vector will typicallydepend on the compatibility of the vector with the host cell into whichthe vector is to be introduced. The vector may also include a selectionmarker such as an antibiotic resistance gene that can be used forselection of suitable transformants. Examples of such resistance genesare well known to those of skill in the art.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified” “variant” or “mutant” refers to a gene or gene product whichdisplays modifications in sequence and or functional properties (i.e.altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

As used herein, underscoring or italicising the name of a gene shallindicate the gene, in contrast to its protein product, which isindicated in the absence of any underscoring or italicising. Forexample, “AGP-1” shall mean the AGP-1 gene, whereas “AGP-1” shallindicate the protein product of the “AGP-1” gene.

2. Transgenic Mammals of the Invention

The invention provides transgenic, non-primate mammals with a drugmetabolism that is more like that of a selected species of primate. In aparticular preferred embodiment, the selected species of primate ishuman and, thus, the transgenic mammal has a drug metabolism that ismore human-like than that of the wild-type animal. Such transgenicanimals have applications that include but are not restricted to drugscreening, preclinical evaluation of drugs and various toxicological andpharmacological studies.

The invention is particularly directed to non-primate transgenic modelsfor expression of polypeptides associated with drug behaviour and/ordrug metabolism. More particularly, the invention provides a transgenicnon-primate mammal for predicting the likely behaviour of a drug in aselected species of primate, wherein the transgenic mammal expresses atleast a portion of a foreign polypeptide that is associated with drugbehaviour and/or metabolism and that is expressed naturally in theselected species of primate or in a primate of a different species orthat otherwise corresponds to the naturally expressed polypeptide. Inone embodiment, a foreign polypeptide is encoded by a nucleotidesequence contained within a transgene, wherein the nucleotide sequencecorresponds to a wild-type gene of the selected species of primate or toa wild-type-like genetic material. The wild-type-like genetic materialmay consist of an entire gene or a cluster of genes or parts thereof. Itmay also consist of a biologically active fragment of a wild-type gene.The transgene may include genomic DNA or cDNA. In a preferredembodiment, the transgenic mammal is characterised by having at leastone human or human-like gene, encoding a drug binding and/or drugmetabolising polypeptide, inserted into its genome. Preferably, thetransgenic mammal includes stable changes to its germ line sequence withstable integration of the transgene in all or a portion of its cells.

The efficacy of a drug is dependent on the amount of drug that reachesthe target tissue and the affinity the compound has for the target.Similarly, the toxicity of a drug depends on the amount of drug or itsmetabolites that reaches vulnerable tissues. Drugs can be administeredby a variety of techniques (e.g. intravenously, intraperitoneally,intramuscularly, orally, subcutaneously), which typically employ acirculatory fluid including, but not limited to, blood, serum,cerebrospinal fluid and lymphatic fluid for drug delivery to the body'stissues. Several drug-metabolising polypeptides exist within circulatoryfluids, which can affect the half-life of both a drug and itsmetabolites. It is often the secondary metabolites and not the drugitself that determines toxicity and can contribute to efficacy.Generally, two types of metabolism can occur: Phase I metabolism usuallyincreases the polarity of the molecule by oxidation, reduction orhydrolysis and Phase II reactions are synthetic in that some conjugationof an endogenous substrate to the drug occurs (e.g. acetylation; seeTable 1 and Gilman et al. [Eds], 1985, The Pharmacological Basis ofTherapeutics. MacMillan Publishing Co., New York). However, speciesdifferences in drug metabolism can occur. For example, in Phase IIreactions, it is generally considered that in rats, glucuronidation ispreferred over sulfation, whereas in the dog and human, sulfation ispreferred, although exceptions occur (Lin and Lu, 1997, Pharmacol. Rev.49: 403-449). A well-known difference in drug metabolism is that despitehydroxylation of amobarbital being a consistent feature amongst humans,dogs, guinea pigs, rats, hamsters and mice, N-glucuronidation appears tobe human-specific, whereas the formation of a diol derivative appears tooccur only in the non-human species studied (Tang et al., 1980, CanadianJ. Physiol. Pharmacol. 58: 1167-1169). Numerous enzymes are involved indrug metabolism and these include several cytochrome p450 (CYP)isoforms, esterases, acetyl-transferases, acetylases,glucuronosyl-transferases, glucuronidases, glutathione S-transferasesand many more (see for example Table 1). Typically, there are structuraldifferences between drug-metabolising polypeptide homologues fromdifferent species, which can affect their drug-metabolising capacities,including their substrate specificity. Thus, in one embodiment, theforeign polypeptide is a drug-metabolising polypeptide. Preferredforeign polypeptides of this type, include polypeptides that facilitateor catalyse a reaction selected from an oxidative reaction including,but not limited to, dealkylation (O- or N-linked), deamination,desulphuration, hydroxylation (aliphatic or aromatic side chains),hydroxylation (N-linked) and sulphoxide derivativisation, a conjugationreaction including, but not limited to, acetylation, glucuronidation,glycine conjugation, methylation (O-, N-, or S-linked) and sulphateconjugation, a hydrolytic reaction including, but not limited to,hydrolysis of esters or amides as well as a reductive metabolismincluding, but not limited to, a reductive metabolism of azo groups ornitro groups. In a particularly preferred embodiment, thedrug-metabolising polypeptide is a Cytochrome P450 (CYP), which issuitably selected from a CYP family including, but not limited to, CYP1, CYP 2, CYP 3 and CYP 4 families. In a preferred embodiment of thistype, the foreign polypeptide is a human or human-like CYP subtype orhaplotype, which is preferably, but not exclusively, selected fromCYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5,CYP4A9 or CYP4A11. The expression of endogenous CYPs may be leftunaltered or disrupted. In an especially preferred embodiment of thistype, the CYP is CYP3A4. In another preferred embodiment, thedrug-metabolising polypeptide is a uridine diphosphoglucuronosyltransferase (UGT).

Other examples of drug-metabolising proteins and genes include membersof the multidrug-resistance (MDR) and multidrug-resistance-associatedprotein (MRP) families. For example, the human MDR-1 gene encodes aP-glycoprotein that acts as a drug-efflux pump, essentially limiting theamount of drug that accumulates intracellularly. MRP-1 has a similar,though distinguishable effect.

Circulatory fluids also contain several proteins (and other factors)that possess binding affinity for certain drugs and that thereby affectthe distribution, efficacy and/or toxicity of these drugs or theirmetabolites. Typically, such drug-binding polypeptides from differentspecies have structural differences, which can affect their drug-bindingcapacities. Thus, in an alternate embodiment, the foreign polypeptide isa drug-binding polypeptide. In a preferred embodiment of this type, thedrug-binding polypeptide is serum albumin, which typically binds acidicdrugs or drug metabolites. Preferably, the foreign polypeptide is humanserum albumin, which suitably comprises the sequence set forth in SEQ IDNO: 2. In a preferred embodiment of this type, the nucleotide sequenceof the transgene, which encodes the human serum albumin, comprises thesequence set forth in any one of SEQ ID NO: 1 and 3. Suitably, theendogenous serum albumin of the transgenic mouse, or ancestor thereof,is a mouse serum albumin comprising the sequence set forth in SEQ ID NO:6. Preferably, the endogenous gene for mouse serum albumin encodes atranscript comprising the sequence set forth in SEQ ID NO: 5. Theregulatory polynucleotide suitably comprises a nucleotide sequence thatis naturally located upstream of the coding sequence relating to theendogenous gene. Preferably, the regulatory polynucleotide comprises thesequence as set forth in SEQ ID NO: 7.

Another drug-binding polypeptide is α-acidic glycoprotein (also known asAAG, AGP, orosomucoid, ORM), which is hereafter referred to as AGP. Theplasma concentration of AGP in healthy individuals ranges from 0.028 to0.092 g/100 mL, and increases in response to inflammation, infection orcancer (Duche J C et al., 1998, J Chromatogr B Biomed Sci Appl 715:03-109; Duche J C et al., 2000, Clin Biochem 33: 197-202; Nakamura H etal., 2000 Biochem Biophys Res Commun 276: 779-784). The level of AGP canvary widely in these disease states and can, therefore, profoundlyaffect the pharmacokinetics of drugs that bind strongly to AGP. Severalsubtypes of AGP have been described and are encoded by 2-3 tandemlyarranged genes, henceforth referred to as AGP-1, AGP-2 and AGP-3. Ingeneral, AGP binds neutral and basic compounds, although exceptions tothis occur (see Lin and Lu, 1997, Pharmacol. Rev. 49: 403-449). Thus, inanother embodiment, the drug-binding polypeptide is an AGP. In apreferred embodiment of this type, the foreign polypeptide is a humanAGP selected from AGP-1, AGP-2 and AGP-3. Suitably, the human AGP-1(also known as orosomucoid (ORM)-1) comprises the sequence set forth inSEQ ID NO: 14. In one embodiment, the nucleotide sequence of thetransgene, which encodes the human AGP-1, comprises the sequence setforth in SEQ ID NO: 13. Suitably, the human AGP-2 (also known as ORM-2)comprises the sequence set forth in SEQ ID NO: 16. In one embodiment,the nucleotide sequence of the transgene, which encodes the human AGP-2,comprises the sequence set forth in SEQ ID NO: 15. In another preferredembodiment, the expression of an endogenous AGP is altered. Suitably,the endogenous AGP is a mouse AGP selected from AGP-1, AGP-2, AGP-3 andAGP-4. Suitably, the mouse AGP-1 comprises the sequence set forth in SEQID NO: 10. Preferably, the endogenous gene encoding the mouse AGP-1comprises the sequence set forth in SEQ ID NO: 9. Suitably, the mouseAGP-3 comprises the sequence set forth in SEQ ID NO: 12. Preferably, theendogenous gene encoding the mouse AGP-3 comprises the sequence setforth in SEQ ID NO: 11. The regulatory polynucleotide, in this instance,suitably comprises a nucleotide sequence that is naturally locatedupstream of the coding sequence relating to the gene encoding theforeign polypeptide. Preferably, the regulatory polynucleotide comprisesthe sequence set forth in SEQ ID NO: 21 and/or 22, which correspond toregulatory polynucleotides located naturally upstream of the human AGP-1and AGP-2 genes, respectively.

Drug-binding proteins may also include the target of a specific drug.For example, the drug Herceptin is a monoclonal antibody (mAb) thatrecognises the extracellular domain of human (but not mouse) ErbB2 andhas the effect of reducing the growth of ErbB2-overexpessing tumours.Since most mAbs are initially generated in mice, it is not surprisingthat many mAbs, such as Herceptin and its precursor 4D5 do not recognisethe homologous mouse protein. Pre-clinical testing of Herceptin includedthe treatment of nude mice bearing human tumours. Such studies did notreveal the potential cardiac toxicity of Herceptin because the ErbB2expressed by the mouse tissues was not recognised by Herceptin. In thecontext of this drug, ErbB2 is considered a drug-binding protein and theinvention, therefore, contemplates any such drug target as a foreignpolypeptide of the invention. In a preferred embodiment, the presentinvention contemplates the humanisation of drug targets such as ErbB2 inthe transgenic mammal.

The transgenic mammal may be produced by standard transgenic (randomintegration) or “knock-in” (site specific) technology and may beassociated with the disruption of a host cell gene, in particular, ahost gene that is homologous, similar or otherwise corresponding to atleast a portion of the transgene. In a preferred embodiment, nucleicacid sequences of the transgene are usually from a human source althoughit may be suitable to derive one or more genes, or parts thereof from anon-human source; for example, a human-like animal such as a non-humanprimate. Alternatively, the transgene may be a hybrid/chimera ofsynthetic polynucleotides and/or human polynucleotides and/orpolynucleotide sequences from other origins.

The transgene of interest is selected for its ability to encode apolypeptide associated with drug binding and/or metabolism. Thesimultaneous use of more than one transgene for insertion into a singleembryo is within the scope of this invention.

Preferably, the transgenic animal is selected from the order Rodentia. Apreferred transgenic mammal is a mouse, although rats are also ofparticular utility. However, it will be understood that the presentinvention is not restricted to these species. For example, thetransgenic animal may be a humanised dog or guinea pig.

Useful sequences for producing the transgenic mammals of the inventioninclude, but are not restricted to, open reading frames encodingspecific polypeptides or domains, introns, and adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation ofexpression. Nucleic acid sequences encoding a polypeptide of interestmay be cDNA or genomic DNA or a fragment thereof.

A genomic sequence of interest comprises a protein-coding region, forexample, as defined in the listed sequences and may include any or allof the introns that are normally present in a native chromosome. It mayfurther include the 3′ and 5′ untranslated regions found in the maturemRNA.

Regulatory polynucleotides including promoters and other regulatoryelements are also used in practising this invention. In someapplications, it is preferable to use regulatory elements from the samespecies as the recipient mammal. In other applications, particularlywhere a pattern of gene expression in the transgenic mammal is requiredto be more like that of the selected species of primate, it may bepreferable to use regulatory elements of that species, or regulatoryelements that are like that species, or a mixture of such regulatoryelements and regulatory elements of the transgenic mammal. Thus, in apreferred embodiment, where a more human-like pattern of gene expressionis required, it may be preferable to use human or human-like regulatoryelements or a mixture of human and host regulatory elements. Preferably,the regulatory elements include genomic sequences, typically but notexclusively of about 1 to about 10 kb in length and corresponding to thesequences upstream of the 5′ and possibly downstream of the 3′ of themRNA encoding region of the host mammalian gene or the correspondinghuman gene to be inserted into the recipient genome. Other regulatoryelements may be located in the introns or exons, including the 5′non-translated sequence, 3′-non-translated sequence and protein codingsequence of a gene. Thus, the regulatory polynucleotide suitablycomprises transcriptional and/or translational and/or otherpost-transcriptional control sequences, which include, but are notlimited to, a promoter sequence, a 5′ non-coding region, acis-regulatory region such as a functional binding site fortranscriptional regulatory protein or translational regulatory protein,an upstream open reading frame, transcriptional start site,translational start site, and/or nucleotide sequence which encodes aleader sequence, termination codon, translational stop site and a 3′non-translated region. A 3′ non-translated sequence refers to thatportion of a gene comprising a DNA segment that contains apolyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression. The polyadenylation signalis characterised by effecting the addition of polyadenylic acid tractsto the 3′ end of the mRNA precursor. Polyadenylation signals arecommonly recognised by the presence of homology to the canonical form 5′AATAAA-3′ and may include T-rich or GT-rich sequences in close proximity(generally 20-60 nt from AATAAA), although variations are not uncommon.The 3′ non-translated regulatory DNA sequence preferably includes fromabout 50 to 1,000 nucleotide base pairs and may contain mRNA cleavagesignals or transcriptional termination sequences as well astranslational termination sequences in addition to a polyadenylationsignal and any other regulatory signals capable of effecting mRNAprocessing or gene expression. Constitutive or inducible promoters asknown in the art are contemplated by the invention. The promoters may beeither naturally occurring promoters, or hybrid promoters that combineelements of more than one promoter. Promoter sequences contemplated bythe present invention may be native to the host cell to be introduced ormay be derived from an alternative source, where the region isfunctional in the host cell. The polynucleotides used in the subjectinvention may encode all or a part of the polypeptides of interest ordomains thereof as appropriate. Fragments of the DNA sequence may beobtained by chemically synthesising oligonucleotides in accordance withconventional methods, by restriction enzyme digestion, by PCRamplification (as for example described in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,965,188 and by Ausubel et al. (“Current Protocols inMolecular Biology”, John Wiley & Sons Inc, 1994-1998) or bymodifications thereof including long range PCR techniques such as LongTemplate PCR System (Boehringer Mannheim, Indianapolis, Ind.; see alsoSkiadas J. et al. 1999, Mammalian Genome 10: 1005-1009) and “inversePCR” (as for example described by Akiyama K. et al. 2000, Nucleic AcidsResearch 28(16)e77 i-vi), or by any other nucleic acid amplificationtechnique such as, but not limited to, strand displacement amplification(SDA) as for example described in U.S. Pat. No. 5,422,252; rollingcircle replication (RCR) as for example described in Liu et al., (1996,J. Am. Chem. Soc. 118:1587-1594 and International application WO92/01813) and Lizardi et al., (International Application WO 97/19193);nucleic acid sequence-based amplification (NASBA) as for exampledescribed by Sooknanan et al., (1994, Biotechniques 17:1077-1080); andQ-β replicase amplification as for example described by Tyagi et al.,(1996, Proc. Natl. Acad. Sci. USA 93: 5395-5400), etc. For the mostpart, DNA fragments will be of at least 10 nucleotides, usually at least18 nucleotides. Such small DNA fragments are useful as primers for PCRor other nucleic acid amplification technique, hybridisation screening,etc. Larger DNA fragments, i.e. greater than 100 nucleotides are usefulfor production of the encoded polypeptide or part thereof. For use inamplification reactions, such as PCR, a pair of primers will be used. Asan example, primers corresponding to regions at the 5′ and 3′ ends of aDNA segment of interest can be chemically synthesised and used in a PCRreaction with genomic DNA or cDNA as the template, in order to generateand amplify the segment of interest.

3. Nucleic Acid Constructs

The invention provides a nucleic acid construct or vector for producinga transgenic non-primate mammal for predicting the likely behaviour of adrug in a selected species of primate. Advantageously, the constructincludes a transgene comprising a nucleotide sequence that encodes atleast a portion of a foreign polypeptide that is associated with drugbehaviour and/or metabolism and that is expressed naturally in theselected species of primate or in a primate of a different species orthat otherwise corresponds to the naturally expressed polypeptide. In anespecially preferred embodiment, the transgene comprises apolynucleotide of human origin or a human-like polynucleotide (e.g.,from a different species of primate) or other equivalent.

In one embodiment, the nucleic acid construct is a targeting vectorcomprising two regions flanking said transgene wherein the regions aresufficiently homologous with portions of the genome of said non-primatemammal to undergo homologous recombination with the portions. In apreferred embodiment of this type, the portions comprise a sequenceflanking, or contained by, the endogenous gene encoding a polypeptide ofthe non-primate mammal, which polypeptide is a corresponding homologueof the foreign polypeptide. The transgene preferably comprises aregulatory polynucleotide operably linked to the sequence that encodesat least a portion of the foreign polypeptide. Suitably, the targetingvector comprises a selectable marker gene.

Thus, targeting vectors for homologous recombination will comprise atleast a portion of the foreign or heterologous gene of interest, andwill include regions of homology to the target locus. DNA vectors forrandom integration need not include regions of homology to mediaterecombination. Conveniently, markers for positive and negative selectionare included. Methods for generating cells having targeted genemodifications through homologous recombination are known in the art. Forvarious techniques for transfecting mammalian cells, see Keown et al.(1990, Methods in Enzymology 185: 527-537).

It is preferred that regions are selected to be of sufficient length andhomology with portions of the genome to permit the homologousrecombination of the transgene into at least one allele of theendogenous gene resident in the chromosomes of the target or recipientnon-primate cell (e.g. ES cells). Preferably, the regions compriseapproximately 1 to 15 kb of DNA homologous to the intended site ofinsertion into the host genome (more than 15 kb or less than 1 kb of theendogenous gene sequences may be employed so long as the amount employedis sufficient to permit homologous recombination into the endogenousgene).

Suitably, the nucleic acid construct comprises a selectable marker gene.In a preferred embodiment, the nucleic acid construct is a targetingvector comprising a selectable marker gene flanked on either side byregions that are sufficiently homologous with portions of the genome ofsaid non-primate mammal to undergo homologous recombination with thoseportions. In one embodiment, the portions of the genome correspond tosequences flanking or within the endogenous gene encoding a polypeptideof the non-primate mammal, which polypeptide is a correspondinghomologue of the foreign polypeptide. In this instance, the targetingvector is adapted to disrupt the endogenous gene.

The nucleic acid construct may contain more than one selectable makergene. The selectable marker is preferably a polynucleotide which encodesan enzymatic activity that confers resistance to an antibiotic or drugupon the cell in which the selectable marker is expressed. Selectablemarkers may be “positive”; positive selectable markers typically aredominant selectable markers, i.e. genes which encode an enzymaticactivity which can be detected in any animal, preferably mammalian, cellor cell line (including ES cells). Examples of dominant selectablemarkers include the bacterial aminoglycoside 3′ phosphotransferase gene(also referred to as the neo gene) which confers resistance to the drugG418 in animal cells, the bacterial hygromycin G phosphotransferase(hyg) gene which confers resistance to the antibiotic hygromycin and thebacterial xanthine-guanine phosphoribosyl transferase gene (alsoreferred to as the gpt gene) which confers the ability to grow in thepresence of mycophenolic acid. Selectable markers may be ‘negative’;negative selectable markers encode an enzymatic activity whoseexpression is cytotoxic to the cell when grown in an appropriateselective medium. For example, the Herpes simplex virus tk (HSV-tk) geneis commonly used as a negative selectable marker. Expression of theHSV-tk gene in cells grown in the presence of gancyclovir or acycloviris cytotoxic; thus, growth of cells in selective medium containinggancyclovir or acyclovir selects against cells capable of expressing afunctional HSV TK enzyme.

More than one selectable marker gene may be employed with a targetingvector. In this instance, the targeting vector preferably contains apositive selectable marker (e.g. the neo gene) within the transgene anda negative selectable marker (e.g. HSV-tk) towards one or more of saidouter regions flanking the transgene. The presence of the positiveselectable marker permits the selection of recipient cells containing anintegrated copy of the targeting vector whether this integrationoccurred at the target site or at a random site. The presence of thenegative selectable marker permits the identification of recipient cellscontaining the targeting vector at the targeted site (i.e. which hasintegrated by virtue of homologous recombination into the target site);cells which survive when grown in medium which selects against theexpression of the negative selectable marker do not contain a copy ofthe negative selectable marker.

The targeting vectors of the present invention are preferably of the“replacement-type”; integration of a replacement-type vector results inthe insertion of a selectable marker into the target gene. Asdemonstrated herein replacement-type targeting vectors may be employedto disrupt a gene resulting in the generation of a null allele (i.e. anallele incapable of expressing a functional protein; null alleles may begenerated by deleting a portion of the coding region, deleting theentire gene, introducing an insertion and/or a frameshift mutation,etc.) or may be used to introduce a modification into a gene or replacepart or all of the gene. This method may be used when the endogenous orwild-type gene of the mammal is to be disrupted.

Alternatively, the targeting vectors may comprise a recombinase system,which allows for the expression of a recombinase that catalyses thegenetic recombination of a transgene. The transgene is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. In an illustrativeembodiment, either the Cre-loxP recombinase system of bacteriophage P1(Lakso et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236; Orban etal., 1992, Proc. Natl. Acad. Sci. USA 89: 6861-6865) or the FLPrecombinase system of Saccharomyces cerevisiae (O'Gorman et al., 1991,Science 251: 1351-1355; PCT publication WO 92/15694) can be used togenerate in vivo site-specific genetic recombination systems. Crerecombinase catalyses the site-specific recombination of an interveningtarget sequence or transgene located between loxP sequences. loxPsequences are 34 base pair nucleotide repeat sequences to which the Crerecombinase binds and are required for Cre recombinase mediated geneticrecombination. The orientation of loxP sequences determines whether theintervening transgene is excised or inverted when Cre recombinase ispresent (Abremski et al., 1984, J. Biol. Chem. 259:1509-1514);catalysing the excision of the transgene when the loxP sequences areoriented as direct repeats and catalyses inversion of the transgene whenloxP sequences are oriented as inverted repeats.

The vectors used in creating the transgenic non-primate mammal of theinvention may also contain other elements useful for optimal functioningof the vector prior to or following its insertion into the recipientnon-primate mammalian cell. These elements are well known to those ofordinary skill in the art and are described, for example, in Sambrook etal., Cold Spring Harbor Laboratory Press, 1989. Preferably, thetransgene components of the vector are assembled within a plasmid vectorsuch as, for example, pBluescript (Stratagene) and then isolated fromthe plasmid DNA, prior to transformation of the target cells.

Vectors used for transforming mammalian embryos are constructed usingmethods well known in the art including without limitation the standardtechniques of restriction endonuclease digestion, ligation, plasmid andDNA and RNA purification, DNA sequencing and the like as described, forexample, in Sambrook, Fritsch and Maniatis, Eds., Molecular: ALaboratory Manual. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1989]).

4. Methods of Producing the Transgenic Mammals of the Invention

The transgenic mammals of the present invention are preferably generatedby introduction of the targeting vectors into embryonal stem (ES) cells.ES cells can be obtained by culturing pre-implantation embryos in vitrounder appropriate conditions (Evans, et al., 1981, Nature 292: 154-156;Bradley, et al., 1984, Nature 309: 255-258; Gossler, et al., 1986, Proc.Natl. Acad. Sci. USA 83: 9065-9069; and Robertson, et al., 1986, Nature322: 445-448). Transgenes can be efficiently introduced into the EScells by DNA transfection using a variety of methods known to the artincluding electroporation, calcium phosphate co-precipitation,protoplast or spheroplast fusion, lipofection and DEAE-dextran-mediatedtransfection. Transgenes may also be introduced into ES cells byretrovirus-mediated transduction or by microinjection. Such transfectedES cells can thereafter colonise an embryo following their introductioninto the blastocoel of a blastocyst-stage embryo and contribute to thegerm line of the resulting chimeric animal. For review, see Jaenisch(1988, Science 240: 1468-1474). Prior to the introduction of transfectedES cells into the blastocoel, the transfected ES cells may be subjectedto various selection protocols to enrich for ES cells which haveintegrated the transgene assuming that the transgene provides a meansfor such selection. Alternatively, the polymerase chain reaction may beused to screen for ES cells which have integrated the transgene. Thistechnique obviates the need for growth of the transfected ES cells underappropriate selective conditions prior to transfer into the blastocoel.

Alternative methods for the generation of transgenic mammals are knownto those skilled in the art. For example, embryonal cells at variousdevelopmental stages can be used to introduce transgenes for theproduction of transgenic mammals. Different methods are used dependingon the stage of development of the embryonal cell. The zygote,particularly at the pronucleal stage (i.e. prior to fusion of the maleand female pronuclei), is a preferred target for micro-injection. In themouse, the male pronucleus reaches the size of approximately 20micrometers in diameter, which allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster, et al., 1985, Proc. Natl. Acad. Sci. USA 82: 4438-4442). As aconsequence, all cells of the transgenic non-primate mammal will carrythe incorporated transgene. This will in general also be reflected inthe efficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbour the transgene. Micro-injectionof zygotes is the preferred method for random incorporation oftransgenes. U.S. Pat. No. 4,873,191 describes a method for themicro-injection of zygotes.

Retroviral infection can also be used to introduce transgenes into anon-primate mammal. The developing non-primate embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Janenich, 1976, Proc. Natl. Acad. Sci.USA 73: 1260-1264). Efficient infection of the blastomeres is obtainedby enzymatic treatment to remove the zona pellucida (Hogan et al., 1986,in Manipulating the Mouse Embryo, Cold Spring Harbour Laboratory Press,Plainview, N.Y.). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner, D. et al., 1985, Proc. Natl. Acad. Sci. USA 82:6927-6931; Van der Putten, et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152). Retroviral infection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart, et al., 1987, EMBO J. 6: 383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner, D.et al., 1982, Nature 298: 623-628). Most of the founders will be mosaicfor the transgene since incorporation occurs only in a subset of cellswhich form the transgenic mammal. Further, the founder may containvarious retroviral insertions of the transgene at different positions inthe genome, which generally will segregate in the offspring. Inaddition, it is also possible to introduce transgenes into the germline,albeit with low efficiency, by intrauterine retroviral infection of themidgestation embryo (Jahner, D. et al., 1982, supra). An additionalmeans of using retroviruses or retroviral vectors to create transgenicmammals known to the art involves the micro-injection of retroviralparticles or mitomycin C-treated cells producing retrovirus into theperivitelline space of fertilised eggs or early embryos (PCTInternational Application Publication No. WO 90/08832) and Haskell andBowen, 1995, Mol. Reprod. Dev. 40: 386).

In selecting lines of any mammalian species to work this invention, theymay be selected for criteria such as embryo yield, pronuclear visibilityin the embryos, reproductive fitness, colour selection of transgenicoffspring or availability of ES cell clones. For example, if transgenicmice are to be produced, lines such as C57/B16 or 129 may be used.

The age of the mammals that are used to obtain embryos and to serve assurrogate hosts is a function of the species used. When mice are used,for example, pre-puberal females are preferred as they yield moreembryos and respond better to hormone injections.

Administration of hormones or other chemical compounds may be necessaryto prepare the female for egg production, mating and/or implantation ofembryos. Usually, a primed female (i.e. one that is producing eggs thatmay fertilised) is mated with a stud male and the resulting fertilisedembryos are removed for introduction of the transgene(s). Alternatively,eggs and sperm may be obtained from suitable females and males and usedfor in vitro fertilisation to produce an embryo suitable forintroduction of the transgene.

Normally, fertilised embryos are incubated in suitable media until thepronuclei appear. At about this time, the exogenous nucleic acidsequence comprising the transgene of interest is introduced into themale or female pronucleus. In some species, such as mice, the malepronuclease is preferred.

Introduction of nucleic acid may be accomplished by any means known inthe art such as, for example, microinjection. Following introduction ofthe nucleic acid into the embryo, the embryo may be incubated in vitrofor varied amounts of time prior to reimplantation into the surrogatehost. One common method is to incubate the embryos in vitro for 1 to 7days and then reimplant them into the surrogate host.

Reimplantation is accomplished using standard methods. Usually thesurrogate host is anaesthetised and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary, and will usually be comparable to or higher than the number ofoffspring the species naturally produces. Transgenic offspring of thesurrogate host may be screened for the presence of the transgene by anysuitable method. Screening may be accomplished by Southern or northernanalysis using a probe that is complementary to at least a portion ofthe transgene (and/or a region flanking the transgene) or by PCR usingprimers complementary to portions of the transgene (and/or a regionflanking the transgene). Western blot analysis using an antibody againstthe protein encoded by the transgene may be employed as an alternativeor additional method for screening.

Alternative or additional methods for evaluating the presence of thetransgene include without limitation suitable biochemical assays such asenzyme and/or immunological assays, histological stains for particularmarkers or enzyme activities and the like.

Progeny of the transgenic mammals may be obtained by mating thetransgenic mammal with a suitable partner or by in vitro fertilisationusing eggs and/or sperm obtained from the transgenic mammal. Where invitro fertilisation is used, the fertilised embryo is implanted into asurrogate host or incubated in vitro or both. Where mating is used toproduce transgenic progeny, the transgenic mammal may be back-crossed toa parental line, otherwise inbred or cross-bred with mammals possessingother desirable genetic characteristics. The progeny may be evaluatedfor the presence of the transgene using methods described above, orother appropriate methods.

Although the foregoing discussion has been made with reference toseveral methods for producing transgenic mammals, it will be understoodthat the present invention is not predicated on, or limited to, any oneof these methods but instead contemplates any suitable means forproducing genetically modified mammals whose germ cells or somatic cellscontain a transgene as broadly described above.

5. Uses of Genetically Modified Mammals

The transgenic mammals of this invention are used in place of, or inaddition to, the standard mammals from which they are derived. A list ofexample techniques is provided below, which describe various uses of thetransgenic mammals of the invention. These techniques includepharmacokinetic assays, pharmacodynamic assays (including measurement ofefficacy), toxicological assays, as well as studies of absorption,distribution, excretion and metabolism. When used in place of standardmammals, the transgenic mammals provide data that are more predictive ofa drug's behaviour in a selected species of primate, particularlyhumans. When used in addition to standard mammals, the differencesbetween transgenic and standard mammals, with respect to drug behaviour,indicate the potential role of the transgene in the metabolism of thespecific drug under study.

The transgenic mammals described in this invention can be used inseveral standard applications in a manner analogous to the use of normalmammals or mammals bearing other genetic modifications. The generaldescriptions below are intended to illustrate the possible use of thetransgenic mammals of the invention and are not intended to limit thescope of the invention. The descriptions are intended to cover severalpossible modifications of the general assays, known to those of skill inthe art.

For example, transgenic mammals may be administered various doses of acompound (possibly bearing a label such as a radioactive isotope orfluorescent group etc) by a variety of possible routes (intravenously(iv), subcutaneously (sc), intraperitoneally (ip), per os (po),intramuscularly (im), intrathecially or other parenteral routes, or byapplication to the skin, mucous membranes or by placing the material inthe feed or drinking water). The numbers of mice per group typicallyrange from 1 to 20, but the experimenter determines the actual number.Compound can be administered once or many times, and mixtures ofcompounds may also be administered either concomitantly or sequentially.Analytical methods described are also not intended to be restricting,but are merely illustrative.

5.1 Pharmacokinetic Assays

Such assays determine the elimination and metabolism of compounds withinthe body of an mammal over a time course. For example, transgenicmammals according to the invention are administered compound and thenblood, or other body fluids or tissues, or excrement (urine or faeces)are collected at various time points following administration.Concentrations of compound(s), or metabolites thereof, are determined byan appropriate analytical method (for example, HPLC usingspectrophotometric determination of analyte). Kinetic data are thentypically analysed by graphical and computational means.

5.2 Pharmacodynamic Assays

Such assays determine the activity of compounds within the body of amammal, normally over a time course. Following administration, theactivity of the compound(s) against the target can either use“whole-body” assays (e.g. blood pressure, respiratory rate,electrocardiogram, electromyogram, neurological activity by measuringelectromagnetic pluses, etc.) or by imaging techniques (e.g. positronemission spectroscopy, nuclear magnetic resonance imaging, echography,etc). Activity of the compound can also be determined by biochemicalmeans. This can include either direct measurement of interaction of thecompound with the target, or by measurement of an upstream or downstreammarker indicative of pharmacodynamic activity. For example, transgenicmammals according to the invention are administered compound and thenblood, or other body fluids or tissues, or excrement (urine or faeces)are collected at various time points following the administration.Direct measurement of target or marker activity in biological samplescan be made by various means (e.g. enzyme assays to determine target ormarker activity, Western blot or ELISA techniques to determine eithertarget abundance and/or activity, Northern blot analysis to determinetarget or marker mRNA levels etc.). Indirect measurement of the targetor marker can include determination of substrate or product levels byvarious analytical methods (e.g. HPLC using spectrophotometricdetermination of analyte). Testing of pharmacodynamic activity can alsoinvolve challenge to the mammal (e.g. artificially raising bloodpressure by chemical or mechanical means, change in diet to promotephysiological changes, surgical intervention to produce a disease state,injection of infectious agents etc.). Pharmacodynamic data are thentypically analysed by graphical and computational means, and are oftencorrelated to compound levels in the tissues. Included inpharmacodynamic studies is the measurement of drug efficacy in thetreatment of disease. For example, the dose required to inhibit tumourgrowth or eliminate infectious agents.

5.3 Absorption, Distribution, Metabolism and Excretion (ADME) Studies

Such studies typically administer radioactive compound(s) to mammals.Determination of the extent of absorption of the compound(s) into thebody can use the approaches described for pharmacokinetic assays.Compound distribution studies determine the distribution of the compoundinto body tissues and typically use autoradiography of whole-bodyhistological-grade slices. Excretion studies elucidate the routes ofcompound elimination from the body by determination of compound ormetabolite levels in faeces or urine (but can also include extractedbody fluids such as bile) as described for pharmacokinetic assays.Metabolism studies determine the nature and quantity of metabolites ofthe parent compound produced by the mammal. It includes metabolitesexcreted from the body or those remaining internally. Metabolitequantification and identification can use a variety of analyticaltechniques (e.g. mass spectrometry).

5.4 Toxicological Assays

Such assays determine the toxic activity of compounds within the body ofan mammal over a time course. These assays normally use escalation ofdose, either concurrently or sequentially, to groups of mice in order todetermine doses where no toxic effects can be observed. These assays canuse single or multiple administrations of compound(s), and can last forprotracted periods of time (typically two weeks to two years). Followingadministration, the toxic activity of the compound(s) can be determinedby monitoring, by visual inspection, the degree of morbidity (e.g.clinical appearance of the mammal) or mortalities produced, measurementof body weight loss or general activity (e.g. movement, exploratoryactivity, sleep time etc.), food and water consumption, appearance ofthe urine and/or faeces. Determination of the toxic activity of thecompound(s) can also use “whole-body” assays or by imaging techniques(described above for pharmacodynamic assays). Toxic activity of thecompound can also be determined by biochemical means (e.g. determinationof gene induction or products of genes associated with generaltoxicological responses, determination of biological metabolitesassociated with responses to toxic insult). Histological examination ofthe body tissues removed at death or at sacrifice of the mammal can alsobe used to monitor for toxic effects, as can haematological changes inthe number or character of circulating cells. Carcinogenicity studiesmonitor the production of neoplasms or genetic damage likely to lead tocancer during compound treatment. Teratogenic studies determine theeffect of compound administration of the development of the foetus.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

EXAMPLES Example 1 Obtaining a Knock-in Mouse in which Human SerumAlbumin Replaces the Endogenous Mouse Serum Albumin Step 1. ObtainingHuman Coding Sequences

The human albumin cDNA sequence (XM_(—)031320) is obtained by PCR of ahuman foetal liver cDNA library, using primers designed from and basedon the sequence set forth in GenBank Accession No. XM_(—)031320 [SEQ IDNO: 3]. For example, the two separate PCR reactions described belowgenerate 5′ and 3′ portions of the gene that overlap across a Bsu36Isite. After digestion with Bsu36I, the two halves are joined by ligationinto an appropriate vector such as pBluescript (Stratagene), with a neogene blunt-end ligated into the SmaI site with the BamH1 site in thepolylinker at the 3′ end of the gene. The following primers can be usedfor performing the PCR reactions:

5′ portion of human ALB cDNA: Forward primer (from start codon,overlapping BstEII site=nt 39-63 of SEQ ID NO: 3); HALBIF=(5′)ATGAAGTGGGTAACCTTTATTTCC (3′) [SEQ ID NO: 26]. Reverse primer (from endof coding region, including stop codon and overlapping Bsu36I site;=reverse complement of nt 1843-1869 of SEQ ID NO: 3; HALB3R=(5′)TTATAAGCCTAAGGCAGCTTGACTTGC (3′) [SEQ ID NO: 27]. The fragment is cutwith BstEII (links to 3′ end of 5′ mouse flanking sequence) and Bsu36I(links to 3′ portion of human ALB gene).

3′ portion of human ALB gene: Forward primer (overlapping stop codon,Bsu361 site and HALB3R, =nt 1855-1885 of SEQ ID NO: 3); HALB5F=(5′)CCTTAGGCTTATAACATCACATTTAAAAGC (3′)) [SEQ ID NO: 28]. Reverse primer (3′of 2^(nd) polyadenylation signal, =reverse complement of nt 2197-2216 ofSEQ ID NO: 3); HALB4R=(5′) AACTTAGAAGAGTATTAATG (3′)) [SEQ ID NO: 29].The fragment is ligated in the appropriate orientation (3′ end nearestthe SpeI site of the vector) into the pGEM-Teasy vector, cloned andexcised with SpeI and Bsu36I. The SpeI site permits cloning into theSpeI site of the pBluescript-based targeting vector and the 5′ BstEIIsite allows ligation to the 3′ end of the 5′ portion of the human cDNAdescribed above.

Additional 3′ sequences that extend beyond the mRNA-encoding region (andmay facilitate efficient polyadenylation) can be obtained by using anextended version of HALB4R that contains additional downstream (genomic)sequences. For example, (5′)TCATAATGTCAAAATATTATTTTGAATGTTTATAATCCATAACTTAGAAGAGTAT TAATG (3′) i.e.reverse complement of nt 18889-18830 of SEQ ID NO: 1.

An alternative approach is the screening of human liver cDNA librariesusing probes based on coding sequences within SEQ ID NO: 3. Full-lengthalbumin coding sequences can then be derived from the positive clonesusing standard molecular biology techniques (e.g., restriction enzymedigestion and/or PCR). Full length cDNA corresponds to human ALB mRNA asset forth in SEQ ID NO: 3.

Step 2. Obtaining the Flanking Mouse Genomic Sequences Option A

The 5′ mouse flanking sequence is shown in SEQ ID NO: 7 and includes themouse albumin promoter that will drive expression of the transgene. Thissequence is obtained by PCR of mouse genomic DNA using as a forwardprimer nt 1-32 of SEQ ID NO: 7 (J04738) i.e., (5′)AAGCTTGAAAACAGGACTGCCTTAGAAGTAAC (3′)) [SEQ ID NO: 30] and as a reverseprimer the reverse complement of nt 2035-2065 from SEQ ID NO: 7 (J04738)i.e., (5′) GTGGGGTTGATAGGAAAGGTGATCTGTGTGC (3′) [SEQ ID NO: 31], (withinthe 5′-UTR of mouse albumin gene).

The 3′ mouse flanking sequence is also generated by PCR of mouse genomicDNA. The forward primer is derived from nt 1973-2002 of SEQ ID NO: 5(AJ011413) i.e., (5′) TTTAAACATTTGACTTCTTGTCTCTGTGCTGC (3′) [SEQ ID NO:32], (corresponding to the 3′-UTR of mouse albumin mRNA). The reverseprimer is the reverse complement of nt 1-29 of SEQ ID NO: 8 (J05246)i.e., (5′) GTGTCTAGAGGTCCAGACATGTTTGCTAA (3′) [SEQ ID NO: 33], (mousealpha fetoprotein [AFP] 5′ promoter region). This corresponds to aregion which lies ˜12.6 kb downstream of the mouse ALB gene. Thus thedescribed PCR reaction will generate an amplicon of ˜12.6 kb thatcorresponds to the region immediately 3′ of the mouse ALB gene. Thisamplicon can be used directly for construction of the targeting vector.Alternatively, the amplicon can be used to derive a smaller 3′ flankingsequence (e.g., by subcloning into a vector such as pBluescript(Stratagene) followed by restriction enzyme digestion). Smaller flankingregions are often preferable when utilising large genomic codingsequences.

Option B

A contig of 15295 bp that spans the first 11 exons of the mouse albumingene (sequence ID c077802366) is schematically illustrated in FIG. 1 andits sequence is set forth in SEQ ID NO: 34. Also included in this contigis about 2 kb upstream of the gene that contains most of the promotersequence. A 5′ arm which spans the mouse promoter region and the 5′UTRof the mouse albumin gene from position 353-2382 on the contig isgenerated by PCR using the primers below and mouse genomic DNA as atemplate. The fragment is ligated in the appropriate orientation (5′ endnearest the SacII site of vector) into the pGEM-Teasy vector, cloned andexcised with NotI and BstEII. The NotI site permits cloning into theNotI site of the pBluescript-based targeting vector and the 3′ BstEIIsite allows ligation to the 5′ end of the human albumin cDNA describedabove. The 5′ flank, together with both halves of the cDNA are ligatedinto the targeting vector cut with NotI and SpeI. The resultantconstruct contains the mouse promoter and 5′ UTR linked to the humancoding region and human 3′UTR. Although the first 3-6 codons are ofmouse origin, these codons are 100% identical in human and mouse albuminmRNA.

Examples of useful primers for the above PCR amplification include:Malb353F (5′) CATATAGGACGAGTGCCCAGGAG (3′) (˜2 kb upstream of startcodon; =nt 353-375 of SEQ ID NO: 34) [SEQ ID NO: 35]; and Malb2382R (5′)GGTTACCCACTTCATTTTGCCAGAGGCTAGTGGGGTTGATAGG (3′) [SEQ ID NO: 36] (whichoverlaps a BstEII site, the start codon and a portion of the 5′UTR; i.e.reverse complement of nt 2354-2397 of SEQ ID NO: 34 with the 5′ end ofthe oligo containing the reverse complement of human sequence nt 78-93of SEQ ID NO: 3).

A 3′ arm (position 6310 to position 13382 in the contig) is generated byPCR of mouse genomic DNA using the following oligos as primers:

Malb630F (5′) CCG62

CGAGTGAAGTTGCCAGAAGACATCC (3′) [SEQ ID NO: 37]; and Malb13382R (5′)ACGCGTCGACAAGAGACGATTCACCCAACC (3′) [SEQ ID NO: 38].

The resultant 7 kb fragment, which extends from the middle of exon 5downstream to near the end of intron 10, is cut with XhoI (site existsin forward primer) and SalI (site exists in reverse primer) and ligatedinto the SalI site of the targeting vector.

Following homologous recombination, this targeting vector is capable ofreplacing mouse ALB exons 1-4, with the human ALB cDNA, which includes apolyadenylation signal that prevents expression of the remainingundeleted portion of the mouse ALB gene (see FIG. 2).

An alternative strategy involves amplifying a fragment downstream of theentire mouse albumin gene thus deleting the whole gene locus. Using a27781 bp mouse contig (Sanger Assembly No. F105491, as set forth in SEQID NO: 39), primers can be designed to amplify a 3′ arm of about 7 kb;e.g. as follows: Reverse primer; albt9649R (5′)AGCTCTCGAGAATCCCTGCCTTTCCTCC (3′) [SEQ ID NO: 40]; and Forward primeralbt2842F (5′) AGTAGTCGACGACAGCAGATGCCT GTGATCC (3′) [SEQ ID NO: 41].

The reverse primer is the reverse complement of nt 9667-9649 from SEQ IDNO: 39. The forward primer is nt 2842-2862 from SEQ ID NO: 39. Theresultant 7 kb fragment, is cut with XhoI (site exists in forwardprimer) and SalI (site exists in reverse primer) and ligated into theSalI site of the targeting vector.

Step 3. Assembling the Transgene Vector

The components obtained in steps 1 and 2 are assembled within a plasmidsuch as pBluescript (Stratagene), in the following order; (5′ mouseflanking)---(human ALB)---(neo)---(3′ mouse flanking). The neomycinresistance gene driven by the TK promoter is blunt-end ligated into theSmaI site of pBluescript with the 3′ end of the gene near the BamHIsite. Where human albumin cDNA is used, the resultant plasmid is thencleaved with NotI/SpeI and ligated with the 5′ arm containing the mousepromoter (5′ NotI-BstEII 3′), the 5′ portion of the human gene(BstEII---Bsu361), and the 3′ portion of the human gene (5′Bsu36I---SpeI). Clones containing the correct sequences are then cutwith SalI and the 3′ arm is ligated in.

Insertion of a negative selection marker such as HSV-tk at the 3′ end ofthe 3′ flanking sequence is optional but assists in distinguishinghomologous recombinants (which lose the HSV-tk) from random integrants(which maintain the HSV-tk and are thus sensitive to gancyclovir).

Step 4. Inserting the Transgene into ES Cells

When the 3′ arm fragment is inserted in the correct orientation, aunique SalI site is preserved at the 3′ end of the construct and can beused to linearise the targeting construct prior to electroporation in EScells. It is then transfected into mouse embryonic stem (ES) cells,which are later selected in growth medium containing G418 to isolatecells that have incorporated the foreign DNA into their nuclear material(for a detailed protocol, see Examples 6 and 7. In the case where theHSV-tk gene is also used, the cells are further selected in gancyclovirto remove cells that integrated the transgene randomly rather than byhomologous recombination. Individual clones are then grown and eachclone is split into multiple plates. Homologous recombinants areconfirmed by Southern blotting or PCR. This is done by screening withexternal Southern blot probes or external PCR primers at both ends ofthe construct to ensure that the construct has been targeted correctlyat both ends.

In an alternative strategy, the neo cassette is flanked by loxP sitesand can be removed by transient transfection of the ES cells with aCre-expression plasmid or removed from subsequent generations of micethrough interbreeding with Cre-expressing transgenic mice. In anotheralternative strategy, the neo cassette is flanked by FRT sites and canbe removed by transient transfection of the ES cells with aFLP-expression plasmid or removed from subsequent generations of micethrough interbreeding with FLP-expressing transgenic mice.

Step 5. Blastocyst Injection

ES cells from one or more correct clones are injected into mouseblastocysts which are then implanted into pseudo-pregnant mice.Implantation is performed on anaesthetised mice using a dissectingmicroscope. A pseudo pregnant female mouse is anaesthetised with 0.017to 0.020 mL/g body weight of avertin injected IP. The mouse is placedunder the dissecting microscope and an incision area is disinfected with70% ethanol. An ovary is exteriorised and the bursal sack that surroundsthe ovary and the oviduct is carefully pulled open. The ovary andoviduct are separated to expose the opening of the oviduct. The mouseblastocyst is loaded into a reimplantation pipette and the tip of thepipette is inserted several millimetres into the infundibulum andemptied into the oviduct. The ovary is then returned to the peritoneumand the body wall and skin is sutured.

Step 6. Selecting Transgenic Mice

Preferably, the ES cells and blastocysts are obtained from differentstrains of mice such that the chimeric founder (F₀) mice can beidentified by coat colour. When mature, F₀ mice are mated with wild typemice to obtain germline transmission of the targeted allele and the F₁mice containing the desired genetic modification are identified bycolour and confirmed by Southern blot and/or PCR and/or DNA sequencing.The heterozygote F₁ mice can be assayed at this stage for expression ofthe human albumin gene by extracting RNA from the liver of these miceand assaying expression by RT-PCR or Northern blot analysis with a humangene specific probe.

Subsequent generations of transgenic mice are preferably bred tohomozygosity to provide mice that express human serum albumin and do notexpress mouse serum albumin.

An alternative strategy would involve the fusing of the mouse promoterlacking the mouse 5′ UTR with the entire human albumin cDNA includingthe human 5′ UTR and the human 3′UTR. Another alternative strategy wouldinvolve fusing the human coding sequence with the 5′UTR and 3′UTR of themouse albumin gene.

In an alternative strategy a targeting vector is built without humanalbumin cDNA sequence. The construct is used for targeting the mousealbumin gene and knocking out expression of the gene. Once targetedcells no longer expressing the mouse albumin are identified, a human BACspanning the albumin locus RP11-580P21 (sequence ID: AC108157), which isobtainable from Children's Hospital Oakland Research Institute (CHORI),is transfected in together with 1/50^(th) amount of a selectable markersuch as the puromycin- or hygromycin-resistance genes. The cells areselected and individual colonies picked, expanded and samples frozen.DNA is prepared from individual clones and screened for presence of thetransgene and copy number. A single copy is preferable. If necessaryinverse PCR can be carried out by standard protocols to identifyintegration site of the transgene and to ensure that the integration hasnot disrupted another gene.

In an alternative strategy, a fragment can be digested out of theabove-mentioned BAC that spans the entire human albumin locus. Apossible enzyme would be Eco47III that digests the BAC about 8 kbupstream of the beginning of the gene and about 2 kb downstream of thegene. This fragment could be isolated by pulse field gel electrophoresis(PFGE) and cloned into a suitable vector with a selectable marker, whichcan then be used for transfection into the ES cells in which the mousealbumin gene has been deleted. An alternative method includes randomintegration of the human albumin gene by embryo injection and may or maynot be combined with disruption of the endogenous mouse serum albumingene.

Example 2A Obtaining a Mouse in which Human AGP Replaces the EndogenousMouse AGP

Knock-Out Mouse Genes by Homologous Recombination, then RandomlyIntegrate Human Genes:

Step 1. Design of a Targeting Construct to Knockout the Entire Mouse AGPLocus

A BAC sequence has been identified that spans the AGP gene locus inmouse. The BAC is mouse chromosome 4 BAC279 (GenBank Accession No.AF336379). The region has been mapped and four genes (or pseudogenes)have been identified. They span a region extending from approximately98880 to 120991 in this sequence. A 41400 bp region spanning fromposition 88681 to position 130080 can be taken from this BAC sequenceand used for preparation of the targeting construct. A linear map ofthis region is shown in FIG. 3 and the sequence relating thereto is setforth in SEQ ID NO: 42.

The mouse 5′ arm and 3′ arm for the targeting vector are obtained fromregions 5′ to the 4 gene locus and 3′ to it, respectively. Suchsequences are obtained by PCR using BAC279 or mouse genomic DNA as atemplate. Examples of useful primers are summarised below:

Oligonucleotides for amplifying 5′ and 3′ arms of AGP locus Start Endposition position RE Oligo name in seq in seq Seq site F = forward idno: id no: Sequence of primer in Id at R = reverse 42 42 the5′-3′ direction no: end AGP99R 9903 9882 TCATTACAACCCCTCTTTAACC 43 SpeIAGP45F 4583 4604 GGACACCAACTACTGACATAGG 44 SpeI AGP49R 4921 4900CCACAGAGATGCTACTGACACC 45 SpeI AGP12F 1227 1245 GCAGAAGGTGAGAAGATGG 46SpeI AGP339F 33991 34010 TCCAAAATGCTTCAGAGACC 47 SalI AGP403R 4035340333 AGTGACCAGAGAGCAGAGACC 48 SalI AGP5′exF 85 106GCTACCTCCCACTGTGAAATCG 49 N/A AGP5′exR 962 943 CACAAGCAGTATGCAGGTGG 50N/A AGP3′exF 40365 40384 AGTCTGGGTACATCCCGAGG 51 N/A AGP3′exR 4098240963 CAGACACATGCCACTCCACC 52 N/A

Primers AGP5′EXF and AGP5′EXR amplify an 877 bp 5′ external probe forscreening targeted clones. Primers AGP3′EXF and AGP3′EXR amplify a 617bp 3′ external probe for screening targeted clones.

In the case of the 5′ arm, two separate PCR reactions are performed;with primers AGP12F and AGP49R to give a 3.7 kb fragment and withprimers AGP45F and AGP99R to give a 5.4 kb fragment. Each fragment isdigested with SpeI and HindIII. The SpeI site is located at the end ofthe oligonucleotides and HindIII is around position 4700. The twoSpeI-HindIII fragments are ligated together in the correct orientationand cloned into the SpeI site of a pBluescript vector in which the neogene has been blunt-end ligated into the SmaI sites, with the 3′ end ofthe gene near the BamHI site. The 5′ arm is 8.7 kb long.

The 3′ arm is amplified with primers AGP339 and AGP403 to give a 6.4 kbfragment with a SalI site at each end. The fragment is digested andcloned into the SalI site of the pBluescript vector described above. Alinear map of the resulting construct is shown in FIG. 4.

Step 2. Targeting the Mouse AGP Locus

As per example 1, step 4.

Step 3. Introducing Human Coding Sequences Option a Introducing EntireBAC Clone

The human AGP genes (ORM1 and ORM2) are located on BAC clone RP11-82I1,which is obtainable from the CalTech human BAC library B SangerSequencing Centre, Cambridge, U.K. [bA82I1, see also GenBank AccessionNo. AL356796]. In one strategy, the entire BAC clone or a large fragmentthereof containing the humanAGP genes is randomly incorporated into thegenome of ES cells, in which the mouse AGP locus has been disrupted byhomologous recombination of the construct described above (without humansequences in targeting vector). The BAC is transfected in together with1/50^(th) amount of a selectable marker such as puromycin or hygromycin,cells are selected and individual colonies picked, expanded and frozen.DNA is prepared from individual clones and screened for presence of thetransgene and copy number. If necessary, inverse PCR can be carried outby standard protocols to identify integration site of the transgene andto ensure that the integration has not disrupted another gene.

Option B Introducing a Portion of the BAC Clone Containing Human AGPGenes

In an alternative strategy, the region of the BAC containing the humanAGP genes and flanking sequences are subcloned into a vector containinga selectable marker other than neo. A portion of RP11-82I1 (nt 48000 to78000) contains the human AGP locus whose sequence is set forth in SEQID NO: 19.

Positions of regions in the two genes are as follows:

AGP-1 AGP-2 ATG 6315 13086 stop codon 9523 16300 end of mRNA (polyA)9647 16424

PCR primers for amplifying the desired fragments can be identified fromthis contig. (SEQ ID NO: 19). Alternatively, useful RE sites for cuttingout the relevant fragments can be obtained from the restriction map ofthis contig. For example XbaI cleaves at positions: 3060, 9883, 16600,29368.

Digesting BAC clone RP11-82I1 with XbaI will give many fragments, butamong them there will be wo fragments of 6823 bp and the other of 6717bp which are the AGP-1 and AGP-2 genes respectively together with around3 kb of upstream sequence. The two fragments are assembled in thecorrect orientation in the XbaI, SpeI or NheI sites of a suitable vectorcontaining an alternative selectable marker to neo. Flanking thesecloning sites (and the selectable marker gene) are unique RE sites (e.g.SgrAI) that facilitate removal of the assembled (13.5 kb) human sequence(containing ˜3 kb of sequence 5′ to the AGP-1 gene).

In an alternative strategy a larger fragment can be subcloned from theBAC containing more 5′ sequence in case the transcriptional regulatoryelements extend further upstream. For example, XhoI cuts at position 307and SpeI cuts at positions 10128 and 16905 as set forth in SEQ ID NO:19. Digesting with XhoI and SpeI will give two fragments that can becloned sequentially into a suitable vector and thus give a 16.6 kbgenomic fragment containing ˜6 kb of sequence 5′ to the AGP-1 gene.

Example 2B Knock-Out Mouse Genes and Insert Human Genes at Same Locus byHomologous Recombination—Procedure 1

The preferred strategy involves incorporating the human AGP sequencesinto a mouse AGP targeting vector, between the 5′ arm and the neo gene.In other words, create a knock-in vector that, upon homologousrecombination, both disrupts expression of the mouse AGP genes and alsoinserts the human AGP genes at the same locus. This strategy requiresconstruction of a large vector. There is evidence in the literature thatlow copy number vectors such as pBR322 are more amenable to the cloningof large fragments of above 20 kb. The low copy number aids stability ofthe relatively large plasmids. In a non-ligation mediated cloningprocedure, it was shown that high copy vectors such ColE1-derivedpBluescript were capable of cloning up to 25 kb whilst pBR322 werecapable of up to 80 kb (Lee et al. 2001, Genomics 73: 56-65).

The 5′ and 3′ arms together with the neo gene and the human genomicfragment are cloned in the correct orientation into a single vector,such as pBR322. To facilitate cloning, it is preferable to reduce thesize of the arms relative to those described in Example 2A.

Step 1. Obtaining Human Coding Sequences

The human AGP gene cluster, containing AGP-1, AGP-2 and both promoterregions is obtained by PCR of either human genomic DNA or the BAC clonecontaining SEQ ID NO: 19, which is available from the CalTech human BAClibrary B and the Sanger Centre, Cambridge, UK and is identified asclone RP11-82I1 (bA82I1). The PCR primers are designed from and based onSEQ ID NO: 19 (complete human AGP gene cluster, derived from GenBankAccession No. AL356796). As with Example 1, the preferred methodinvolves two separate PCR reactions to generate the 5′ and 3′ halves ofthe desired human sequence, and then joining the two fragments.Appropriate primers include:

5′ half (AGP1): Forward primer (˜6 kb upstream of AGP-1 translationstart codon), =nt 47283-47314 of SEQ ID NO: 19 (AL356796). i.e. (5′)CAGGCTGCGCCTGGG ATCTCTACACTCGAGCA (3′) [SEQ ID NO: 53]. Reverse primer(3′ of AGP-1 polyadenylation signal, =reverse complement of nt58112-58142 of SEQ ID NO: 19 [AL356796]); i.e. (5′)CTGCACATACGGAATAGATGGAACAACTCAG (3′) SEQ ID NO: 54].3′ half (AGP2): Forward primer=nt 58112-58142 of SEQ ID NO: 19[AL356796]); i.e. (5′) CTGAGTTGTTCCATCTATTCCGTATGTGCAG (3′) SEQ ID NO:55]. (2 kb upstream of AGP2 translation start codon). Reverse primer (3′of AGP2 polyadenylation signal, =reverse complement of nt 66131-66157 ofSEQ ID NO: 19 [AL356796]); i.e. (5′) CCTTTGCCTATCTCAGAACCATAAATC (3′)SEQ ID NO: 56].

The 5′ and 3′ halves obtained by PCR can be spliced together to generatethe human AGP-1-AGP-2 transgene (SEQ ID NO: 20) for ‘knocking in’ to themouse genome.

An alternative strategy involves obtaining the human AGP genes byrestriction enzyme digestion of BAC clone RP11-82I1 (bA82I1) and usingit in the subsequent steps. Alternatively, the entire BAC clone ismicroinjected into mouse embryos (random integration) and the resultanttransgenic mice are later bred with AGP knock-out mice generated viahomologous recombination. For more details on this type of 2-stepprocedure, see Example 3.

Step 2. Obtaining the Flanking Mouse Genomic Sequences

In this example, the transgene contains the human promoters that willdrive expression of human AGP. Therefore, it is not necessary for thehuman polynucleotide sequences to be functionally combined with a mousepromoter as in Example 1. However, it is still preferable tofunctionally inactivate the mouse AGP genes. Therefore, the 5′ flankingmouse sequence is comprised of a 5′ portion of mouse AGP-1 and the 3′flanking mouse sequence is comprised of the 3′ portion of mouse AGP-3,such that homologous recombination results in deletion of the entiremouse AGP-2 gene, the 3′ portion of mouse AGP-1 and the 5′ portion ofmouse AGP-3. The introduction of small mutations into the promoterand/or first exon of mouse AGP1, ensures the functional disruption ofthis gene.

The 5′ mouse flanking sequence is obtainable by PCR of mouse genomicDNA, using as a forward primer, nt 84-111 of SEQ ID NO: 9 (M17376; 5′flank of AGP-1) i.e. (5′) CTACATTTTCAACTCAGATTCACCCCTC (3′) [SEQ ID NO:57]. The reverse primer is the reverse complement of nt 2991-3020 in SEQID NO: 9 (M17376; intron 5 of AGP-1) i.e. (5′)ATGGCTGCTGGCATGTCTGTATGGCAGGCC (3′) [SEQ ID NO: 58]. The resultant PCRproduct, which represents nt. 84-3020 of SEQ ID NO: 9, may then bemutated at critical sites using standard techniques, in order to ensurethat a functional, though truncated, mouse AGP-1 protein is notproduced. The mutations include any or all of the following; a)disruption of the GC box (nt 546-554 of SEQ ID NO: 9) and TATA box (nt562-567 of SEQ ID NO: 9); b) point mutation of the translation startcodon (nt 634-636 of SEQ ID NO: 9) and preferably also the ATG at nt658-660 of SEQ ID NO: 9; c) introduction of a stop codon in the firstexon, such as by changing nt 680 of SEQ ID NO: 9 from T to A. An exampleof a method for introducing mutations is “Fusion PCR”, which utilisesprimers with the desired nucleotide mismatches or 5′ tails encoding themutation. Complementary forward and reverse primers covering themutation are generated and used in separate PCR reactions. One reactioncontains the 5′ forward primer (nt 84-111 of SEQ ID NO: 9 in thisexample) and the mutant reverse primer to generate the mutation as wellas sequences 5′ of it. The other reaction contains the mutant forwardprimer and the 3′ reverse primer (nt 2991-3020 of SEQ ID NO: 9 in thisexample), to generate the mutation as well as sequences 3′ of it. Thetwo PCR products are then combined in a third reaction using the 5′forward and 3′ reverse primers. During annealing, the two different PCRproducts anneal to each other at their complementary ends comprising themutation, while the forward and reverse primers bind the outer ends ofthe same hybrid such that a full-length polynucleotide containing thedesired mutation is synthesised and subsequently amplified.

The 3′ mouse flanking sequence is also generated by PCR of mouse genomicDNA. The forward primer is derived from nt 898-924 of SEQ ID NO: 11([S38219] intron 1 of mouse AGP-3), i.e. (5′)TCATCGTGGATGAATGCCAAGGTCCTC (3′) [SEQ ID NO: 59]. The reverse primer isthe reverse complement of nt 3492-3523 of SEQ ID NO: 11 ([S38219],intron 5 of mouse AGP-3), i.e. (5′) CAAGGTAGGTAAGCCTGTGGGGCAG CTTGAAG(3′) [SEQ ID NO: 60].

Step 3. Assembling the Transgene Vector

The components obtained in steps 1 and 2 are assembled within a plasmidsuch as pBR322, in the following order; (5′ mouse flanking)---(humanAGP-1)---(human AGP-2)---(neo)---(3′ mouse flanking). The techniquesinvolved are well known to those skilled in the art and includerestriction enzyme digestion, ligation and cloning. Unique restrictionenzyme recognition sites added to the 5′ ends of the primers describedabove, are useful for facilitating this procedure.

Insertion of a negative selection marker such as HSV-tk at the 5′ end ofthe 5′ flanking sequence or the 3′ end of the 3′ flanking sequence isoptional but assists in distinguishing homologous recombinants (whichlose the HSV-tk) from random integrants (which maintain the HSV-tk andare thus sensitive to gancyclovir).

Steps 4-6.

As per Example 1.

Example 2C Obtaining a Knock-in Mouse in which Human AGP Replaces theEndogenous Mouse AGP Knock-Out Mouse Genes and Insert Human Genes atSame Locus by Homologous Recombination—Procedure 2 Step 1. Obtaining andAssembling the Flanking Mouse Genomic Sequences

The 8.7 kb 5′ arm (described in Example 2A, above) is digested withSpeI-XbaI to yield a 5.9 kb fragment that can be cloned into the NheIsite of pBR322 or some other vector which contains or lacks the HSV-TKgene. Insertion of a negative selection marker such as HSV-tk at the 5′end of the 5′ flanking sequence or the 3′ end of the 3′ flankingsequence is optional but assists in distinguishing homologousrecombinants (which lose the HSV-tk) from random integrants (whichmaintain the HSV-tk and are thus sensitive to gancyclovir).

The 3′ arm (described in Example 2A, above) is cut with XhoI-SalI toyield a 5072 bp fragment that is cloned into the SalI site of pBR322.The total length of homology arms is almost 11 kb. Between these twoarms lies a unique SgrA1 site that can be used for cloning in the humansequence (see below). Alternatively, artificial primers can be designedand inserted within the SgrA1 site to create a unique NotI site or someother unique RE site(s) for the same purpose.

Step 2. Obtaining and Assembling the Human Genomic Sequences

The 13.5 kb or 16.6 kb human sequence described above is ligated into apBluescript vector, with a modified multi-cloning site (MCS). Examplesof a useful MCS include:

SacI---SgrAI---XhoI---NheI---XbaI---SpeI---BamHI---PstI---SgrAI---KpnI;or SacI---SgrAI---NheI---XbaI---SpeI---neo---SgrAI---KpnI

Such MCSs can be generated by hybridising and ligating synthetic primersor by PCR with extended primers containing the appropriate RE sites.SacI and KpnI allow insertion of the MCS into pBluescript. BamHI/PstIallow insertion of the neo gene. SpeI, XbaI and/or NheI allow insertionof the human 6823 bp and 6717 bp XbaI fragments of RP11-82I1 (containingthe human AGP genes) described in Example 2A above. For example, the 5′fragment is ligated into NheI/XbaI cut vector and clones with the 5′ endat the NheI site identified and cut with XbaI/SpeI, into which the 3′fragment is then ligated. XhoI/SpeI allow insertion of the 16.6 kb humanfragment described in Example 2A. For example, the 9821 bp XhoI/SpeIfragment of RP11-82I1 is ligated into the vector cut with the sameenzymes. The resultant vector is then cut with SpeI and the 6777 bp SpeIfragment of RP11-82I1 inserted to assemble the complete 16.6 kb humanAGP sequence. SgrAI allows removal of the human sequences (with orwithout neo) and subsequent insertion into the pBR322-based targetingvector described above.

It is also possible to make use of a neo gene that not only has aeukaryotic promoter for G418 resistance in ES cells but also contains aprokaryotic promoter that affords kanamycin resistance in E. coli. Whenthe final ligation step is carried out, selection for both the plasmidconferring ampicillin resistance and for the insert conferring kanamycinresistance will aid in selecting for such a large clone. Otherparameters which might aid the construction of a plasmid of close to 30kb in size is the use of high efficiency electrocompetent cells thatsupport the stable propagation of large plasmids such as DH10B andgrowing the clones at reduced antibiotic concentration or at lowertemperature.

An alternative strategy involves two steps of homologous recombination.For example, the initial targeting vector may comprise the 6.8 kb humanfragment containing the human AGP-1 gene. A second round of targetingcould then be carried out using a different selectable marker such ashygomycin or puromycin. The 5′ arm could be all or part of the 6.8 kbhuman AGP-1 gene that had been knocked in during the first targetingevent and the 3′ arm could be the same 3′ arm used in the firsttargeting construct. Between the 5′ arm and the antibiotic resistancegene would be inserted the 6.7 kb human AGP-2 fragment. In such a way,the two genes would be knocked in sequentially during two rounds oftargeting.

Other alternative cloning strategies include ET cloning (Copeland etal., 2001, Nature Reviews Genetics 2: 769-779) or using a cosmid vectoror a bacmid vector to facilitate cloning of the larger human sequences.

Step 3. Targeting the Mouse AGP Locus

As per example 1, step 4.

Step 4. Blastocyst Injection

As per Example 1, step 5.

Step 5. Selecting Transgenic Mice

As per Example 1, step 6.

Example 2D Obtaining a Knock-in Mouse in which Human AGP Replaces theEndogenous Mouse AGP Knock-Out Mouse Genes and Insert Human Genes atSame Locus Using Recombinase Technology Step 1. Design of a TargetingConstruct to Knockout the Entire Mouse AGP Locus

The targeting vector is constructed in a fashion identical to thatdescribed in Example 2A, except that the neomycin resistance gene isflanked at one end with a wild-type loxP site and at the other end witha mutant loxP511 site.

Step 2. Obtaining and Assembling the Human Genomic Sequences

The human gene fragment (16.6 kb or 13.5 kb) is cloned into a separatevector as described in Example 2C, except that the two different loxPsites are incorporated into the modified MCS, immediately adjacent toand between the SgrAI sites, such that they flank the human sequence, inthe correct orientation. There would be a wild-type loxP recombinationsite at one end of the fragment and a mutant loxP recombination site(loxP511) at the other end. These two loxP sites cannot recombinetogether but the loxP511 mutant site can recombine with another loxP511site.

Step 3. Targeting the Mouse AGP Locus

As per Example 1, using the targeting vector described in step 1 (above)in order to disrupt the mouse AGP locus and insert the loxP sites. Oncetargeted ES cells are identified, the human fragment can beco-transfected with the Cre recombinase and in a very efficientrecombination reaction, the neo gene will be replaced by the human genevia recombination mediated Cre excision (RMCE). In such a way it ispossible to efficiently target a single copy of the human genespecifically to the mouse AGP locus, thus controlling both for copynumber and for site of integration. No other gene will be disruptedusing this technique and the AGP gene will be in its “natural locus”eliminating problems of position effects.

An alternative strategy utilises FLP recombinase rather than Crerecombinase. The procedure is identical to that described above, exceptthat;

in steps 1 and 2, a wild type and a mutant FRT site are substituted forthe loxP sites (1998, Biochemistry 37(18): 6229-34); and

in step 3, a FLP-expression plasmid is substituted for theCre-expression plasmid.

Example 3 Obtaining a Transgenic Mouse Expressing Human CYP450 Isoforms

A variety of approaches can be used to generate these transgenic mice.Suitably, polynucleotides encoding one or more human CYP isoformsselected from CYP1, CYP2 and CYP3 families are employed. For example,CYP isoforms from the following list are used as components of thetransgenes: 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, 4A9, 4A11.Desirably, the transgenic mouse will express CYP3A4 and CYP2D6 andespecially it will also express others from this list including CYP2C9and CYP2C19. This may be achieved by incorporating multiple CYP genesinto the same transgenic vector or by the interbreeding of differenttransgenic mice, each containing a different human CYP isoform in orderto generate double, triple quadruple etc. transgenics. Such transgenicsmay be produced by random integration of the transgene or homologousintegration into a specific site and may or may not be associated withdisruption of an endogenous mouse CYP isoform.

Step 1. Obtaining a Polynucleotide Encoding CYP3A4.

The preferred technique involves obtaining a BAC or YAC clone,containing human CYP3A4 and either using the clone directly orlinearising it prior to embryo injection. This technique has theadvantage that multiple CYP3A genes located within the same vector aresimultaneously introduced. Clones RP11-316A24 and RP11-757A13 (GenomeSequencing Center, Washington, U.S.A.) are suitable clones. Otherappropriate BAC or YAC clones are obtained by screening human genomiclibraries using probes designed from SEQ ID NO: 23 relating to BAC cloneRP11-757A13 (GenBank Accession No. AC069294). Reference also may be madeto the sequence set forth in GenBank Accession No. AF209389, whichdefines a 26502 nt polynucleotide comprising exons 1 through 13 of thehuman CYP3A4 gene. AQ539660 and AQ539659 define the BAC ends ofRP11-316A24 and NG_(—)000004 defines the relevant contig.

An alternative strategy involves synthesizing the following primers andwhere appropriate, also including restriction enzyme recognition sitesat the 5′ ends:

Primer 1—(5′) GTCCAAACACTTCTCTATGATAATGCAAACAGTCAC (3′) [SEQ ID NO: 61]i.e. nt 26930-26965 of SEQ ID NO: 23 (AC069294), which is ˜8.4 kbupstream of START codon;

Primer 2—(5′) GTTGCTCTTTGCTGGGCTATGTGCATGG (3′) [SEQ ID NO: 62] i.e. thereverse complement of nt 35227-35254 of SEQ ID NO: 23 (AC069294) and nt26-53 of SEQ ID NO: 24 relating to GenBank Accession No. NM_(—)017460,which is within 5′-UTR of CYP3A4;

Primer 3—(5′) ACAGAGCTGAAAGGAAGACTCAGAGGA (3′) [SEQ ID NO: 63] i.e. nt35255-35281 of SEQ ID NO: 23 (AC069294) and nt 54-80 of SEQ ID NO: 24(NM_(—)017460), which is within 5′-UTR; immediately downstream of Primer2 locus; and

Primer 4—(5′) GACCAATCGACTGTTTTTTATTAAGTG (3′) [SEQ ID NO: 64] i.e.reverse complement of nt 62380-62406 of SEQ ID NO: 23 (AC069294) and nt2738-2764 of SEQ ID NO: 24 (NM_(—)017460), which is within 3′-UTR andincluding poly-adenylation site. Alternatively, an oligo-dT primer,which binds polyA tails, can be used in place of primer 4.

The following PCR reactions are then performed:

PCR Reaction A; Long range PCR is performed using Primers 1 and 2, withBAC clone RP11-757A13 or human genomic DNA as the template. The productcorresponds to ˜8.4 kb of 5′ flanking sequence (including the humanCYP3A4 promoter and enhancers) as well as part of the 5′-UTR.

PCR Reaction B; PCR using Primer 3 and Primer 4, with human liver cDNAas the template. The product corresponds to the entire protein codingsequence as well as the 3′-UTR (including polyadenylation signal) andthe portion of the 5′-UTR not amplified in Reaction A.

Step 2. Assembling the Transgene Vector

The two PCR products are assembled in a suitable vector such aspBluescript (Stratagene). The inclusion of appropriate restrictionenzyme sites at the 5′ end of the primers facilitates this process.

In the case of using a BAC or YAC clone, no assemblage is required.

Step 3. Inserting the Transgene into Mouse Embryos

The BAC clone is purified directly, or linearised and then purified. ThePCR-generated constructs are cut from the plasmid vector and isolatedfrom the plasmid DNA. The purified transgene is then microinjected intothe male pronucleus of isolated mouse embryos, which are then implantedinto the oviduct of pseudopregnant surrogate host mice according tostandard techniques for generating transgenic mice by randomintegration. Additional details are given elsewhere in this document.Alternatively, the transgene DNA is transfected into ES cells (with orwithout a co-transfected selection marker such as neo). Clones arescreened for integration and copy number by Southern blot and/or PCR.

Step 4. Selecting Transgenic Mice

Transgenic mice are identified by PCR and/or Southern blotting andexpression of the transgene is confirmed by Western blotting, ELISA orother immunological assays using appropriate tissue samples, such asliver. Alternatively liver RNA is analysed by Northern blotting.

In another strategy, homologous recombination of the transgene is usedto insert a single copy of the transgene at a desired site in the mousegenome. Preferably, that site is near to or at the site of an endogenousmouse CYP gene similar to the human transgene. For example, mouseCYP3A11, 3A13, CYP3A25 or 3A16, when the human transgene is also amember of the CYP3A family, such as CYP3A4. Flanking mouse sequences areincorporated into the transgene construct as described in Examples 1 and2, in order to achieve homologous recombination. These flankingsequences are, for example, derived from mouse genomic sequences withinor flanking the mouse CYP gene, in the case where it is preferable todisrupt expression of the mouse protein. In the case of transgenic micegenerated by microinjection of BAC or YAC clones, disruption of mouseCYP isoforms is performed in separate mice, which are subsequently bredwith the human CYP-expressing mice described above. A similar techniquehas been used to generate mice expressing human-like but not mouse-likeimmunoglobulins (Green LL. 1999, J. Immunol. Methods 231: 11-23).

The mouse homologues to the human CYP3A genes are located on chromosome5. On this chromosome, there are several CYP3A genes

a) CP3G CYP3A16 (ensembl ID: ENSMUSG0000029628)

b) CP3B CYP3A11 (ensembl ID: ENSMUSG0000029630)

c) CP3P CYP3A25 (ensembl ID: ENSMUSG0000029631)

d) CP3D CYP3A13 (ensembl ID ENSMUSG0000029727)

The order is CYP3A13-˜8 Mb-CYP3A16-˜300 kb-CYP3A11-˜400 kb-CYP3A25.

It is possible to knockout each gene individually. It is also possiblethat in the case of 16, 11 and 25, to carry out two targeting events totarget a wild-type loxP site to the 5′ flanking region and to target amutant loxP511 to the 3′ flanking. Then one could take a human BAC, suchas RP11-316A24, which contains the wt and mutant loxP site at either endand target it to this chromosomal region (with Cre recombinase), thusdeleting all three mouse genes and inserting human CYP3A4, 3A5, 3A7 and3A3, which are contained within this BAC.

Example 4 Obtaining a Transgenic Mouse Expressing Human UGT (UDPGT)Isoforms

The UGT gene family includes UGT1 and UGT2 subtypes. In humans, 9 UGT1genes are known (plus 4 pseudogenes), all of which map to the same locusat 7q22 (Gong et al., 2001; Pharmacogenetics 11: 357-368). Each of thesegenes utilises a unique promoter and exon 1, which encodes substratespecificity, whereas all of these genes share the same exons 2-5, whichencode an identical carboxyl terminus required for interactions withUDP-glucuronic acid.

In the mouse, there are at least 3 genes of the UGT1 family that map tomouse chromosome 1. They are: UD16, UD12 and UD17 which span 86.17 kb,16.65 kb and 5.19 kb of genomic DNA respectively. It is possible thatother genes also lie in this region (including UD11). Each gene has adifferent promoter and exon 1 but they all share the last three exons.These mouse UGT genes have been mapped to a 75 kb contig set forth inSEQ ID NO: 65. The last three exons all map to a 5 kb region.

The position of various exons of each gene on the 75 kb contig are asfollows:

UD17 UD12 Exon 1  1-928 34926-34000 Exon 1  1-952 46298-45347 Exon 2 928-1083 31201-31044 Exon 2  953-1104 31201-31044 Exon 3 1081-130030788-30568 Exon 3 1105-1323 30788-30568 Exon 4 1300-1593 28800-28504Exon 4 1324-1617 28800-28504 UD16 Exon 1-3 out of contig range Exon 4853-922 34128-34059 Exon 5  921-1077 31201-31044 Exon 6 1078-129530788-30568 Exon 7 1294-1587 28800-28504An additional gene also lies on this contig

DNAjb3 Exon 1-1014 41187-42200

Upon homologous recombination, the targeting construct described belowwill delete the last three exons of these genes (and will also deleteexon 1 of UD17 and exon 4 of UD16). Stop codons are inserted in allthree reading frames to ensure that the genes are not expressed. The 3′arm can be amplified using mouse genomic DNA and the following primers:

ugt23275F [SEQ ID NO: 66] (5′) GAAGTCGACGTTTCAGAGTCATACCAAAAGG (3′)(from nt 23275 to nt 23296 in SEQ ID NO: 65); and ugt27501R [SEQ ID NO:67] (5′) GAAGTCGACATCTTACACAGGTCCCAAAGC (3′) (from nt 27501 to nt 27481in SEQ ID NO: 65)

These have artificial SalI sites at the ends. The PCR fragment isdigested with SalI and cloned into the SalI site of the targeting vector(pBluescript with neo gene as described in previous examples).

The 5′ arm can be amplified using mouse genomic DNA and the followingprimers:

ugt35967R (5′) CTAAGAATGAGCAAAGTGTCC (3′) [SEQ ID NO: 68] (from nt 35967to nt 35947 in SEQ ID NO: 65); and ugt31170F (5′)GCAATACTAGCTAGAAAGGCCAG (3′) [SEQ ID NO: 69] (from nt 31170 to nt 31193in SEQ ID NO: 65)

The PCR fragment is cloned into the pGEMTeasy vector, digested with NotIand cloned into the NotI site at the 3′ end of the neo gene. The neogene will be transcribed in the opposite direction of the UGT. The 3′end of this arm contains the first 7-bp of the third last exon (in thecase of UD12 and UD17, exon 3). The neomycin resistance gene willcontain stop codons in all three (antisense) frames for inducingnonsense-mediated decay of the truncated mouse UGT mRNA and terminatingthe translation of any possible mutant UGT protein. It is also possibleto engineer into the targeting vector a fragment containing RNAdestabilising elements to further ensure that any truncated mouse UGTmRNA is degraded rapidly.

The targeting vector will be electroporated into ES cells as describedin Example 6 and ES cell clones in which the region has been correctlytargeted will be picked according to Example 7 and identified bySouthern blot of G418-resistant clones. The targeted ES cells are thentransfected with human BAC clone RP11-943B 10, available from Children'sHospital Oakland Research Institute (CHORI); (BAC ends identified asAQ564938 and AQ711093; Locus defined in AF297093). The BAC may beco-transfected with a plasmid conferring hygromycin or puromycinresistance to assist selection of positive clones. Selected clones arethen expanded and screened for copy number and site of integration asmentioned above. This BAC contains the following human UGT1 genes:UGT1A1, UGT1A2p, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A9,UGT1A10, UGT1A13p (p indicates a pseudogene).

An alternative approach involves using a targeting vector that has theneomycin resistance gene flanked by a wildtype loxP site and a mutantloxP site (loxP511). Cells targeted with this vector can be transfectedwith the BAC, which has the wild type and mutant loxP sites in itsvector backbone together with the a plasmid expressing the CRErecombinase. Colonies that have undergone recombination-mediated Creexcision (RMCE) are selected. In such clones, the neo gene (at the mouseUGT locus) is replaced by the human UGT genes. Alternatively, a wildtypeand a mutant FRT site can be used in conjunction with the FLPrecombinase.

Example 5 Obtaining a Transgenic Mouse Expressing Human MDR-1

The human MDR-1 gene encodes a large transmembrane protein(P-glycoprotein or P-gp) expressed in a variety of tissues includingliver, kidney and intestinal epithelium. P-gp is an integral part of theblood-brain barrier and functions as a drug-transport pump transportinga variety of drugs from the brain back into the blood. Indeed, the MDR-1P-glycoprotein extrudes a variety of drugs across the plasma membrane ofmany cell types.

Whereas humans have only one MDR-1 gene, mice have two. MDR-1a is highlyexpressed in the intestinal epithelium, where it actively excretesxenobiotics absorbed from the intestinal lumen and at the blood-brainbarrier, where it protects the brain from xenobiotics in the blood.MDR-1b is highly expressed in the adrenal gland, pregnant uterus andovaries. Both mouse genes are substantially expressed in many othertissues.

Naturally occurring Mdr-1a mutant mice (of the CF-1 outbred mousestrain) that lack the Mdr-1a P-glycoprotein have been described(Umbenhauer et al., 1997, Toxicol. Appl. Pharmacol. 146(1): 88-94).Knock-out mice have also been generated for each mouse mdr1 gene aloneand in combination (Shinkel et al., 1997; Proc. Natl. Acad. Sci. USA 94:4028-4033). Transgenic mice expressing human MDR-1 can therefore be bredwith any of the above-mentioned mice to generate mice that express thehuman but not mouse forms of these genes.

The following example describes a method for constructing transgenicmice expressing human MDR-1. The human MDR-1 cDNA (GenBank Accession No.NM_(—)000927) [SEQ ID NO: 70] is obtained by RT-PCR of human liver mRNAand assembled in a vector together with human 5′ and (preferably) 3′flanking regulatory sequences and a selectable marker gene. Flankingsequences are obtained by PCR of human genomic DNA or BAC Clone CTB60P12(GenBank Accession No. AC002457) for the 5′ flank and BAC CloneCTB137N13 (GenBank Accession No. AC005068) for the 3′ flank, each ofwhich is obtainable from CalTech human BAC library B. The assembledtransgene is then introduced by random integration as described inExample 6 and previous examples.

The following primers are useful for this purpose. Enzyme sites added tothe 5′ ends are indicated and should be flanked by sufficientnucleotides to permit efficient digestion:

Primer MDR1 (forward) (5′) SalI-GAAACCCTAGGCACTAAATCCC (3′) [SEQ ID NO:71] which overlaps AvrII site in promoter; nt 4930-4951 in 5′ flankhuman MDR contig set forth in SEQ ID NO: 72.

Primer MDR2 (reverse) (5′) ClaI-GGGATTTAGTGCCTAGGGTTTC (3′) [SEQ ID NO:73], which overlaps AvrII site in promoter; reverse complement of nt4930-4951 of 5′ flank human MDR contig [SEQ ID NO: 72].

Primer MDR3 (forward) (5′) CTCATTCTCCTAGGAGTACTCAC (3′) [SEQ ID NO: 74],which overlaps AvrII site in exon 1; nt 10049-10071 of 5′ flank MDRcontig [SEQ ID NO: 72] and nt 56-72 of MDR-1 cDNA [SEQ ID NO: 70].

Primer MDR4 (reverse) (5′) GTGAGTACTCCTAGGAGAATGAG (3′) [SEQ ID NO: 75],which overlaps AvrII site in exon 1; reverse complement of nt10049-10071 of 5′ flank MDR contig set forth in SEQ ID NO: 72 and nt56-72 of MDR-1 cDNA [SEQ ID NO: 70]

Primer MDR5 (forward) (5′) SalI-AAAGCTTGCAGTGTAAGATGCG (3′) [SEQ ID NO:76]; nt 292-313 of 5′ flank MDR contig [SEQ ID NO: 72].

Primer MDR6 (reverse) (5′) ClaI-CACATGAAAGTTTAGTTTTATTATAGAC AC (3′)[SEQ ID NO: 77]; reverse complement of nt 4614-4643 of MDR-1 cDNA [SEQID NO: 70] (in 3′UTR, downstream of PmeI site).

Primer MDR7 (reverse) (5′) XbaI-TGGTCAACAGAGCAAGACTCCGCTTC (3′) [SEQ IDNO: 78], which is located ˜1730 bp downstream of the MDR1polyadenylation signal; nt 36075-36100 of GenBank Accession No. AC005068(BAC Clone CTB137N13, which is obtainable from CalTech human BAC libraryB). A 1940 nt sequence corresponding to reverse complement of nt36061-38001 of GenBank Accession No. AC005068 is presented in SEQ ID NO:79. Primer MDR7 is the reverse complement of nt 1900-1926 in thissequence.

Primer MDR8 (forward) (5′) GCGCCAGTGAACTCTGACTGTATGAGATG (3′) [SEQ IDNO: 80]; nt 4255-4283 of human MDR-1 cDNA [SEQ ID NO: 70], which islocated in 3′ UTR upstream of the PmeI site.

Obtaining Human 5′ Flank with Promoter ElementsPCR Reaction 1: Production of a SaLI-ClaI Fragment Extending from 5 kbto 10 kb Upstream of Transcription Start Site.

PCR is performed with primers MDR5 and MDR2, using as a template, humangenomic DNA or more preferably, DNA from a BAC clone such as CTB-60P12(obtainable from CalTech human BAC library B; GenBank accession numberAC002457), which contains the appropriate sequence.

PCR Reaction 2: Production of an AvrII Fragment Extending from withinExon 1 to ˜5 kb Upstream of Transcription Start Site.

PCR is performed with primers MDR1 and MDR4, using as a template, humangenomic DNA or more preferably, DNA from a BAC clone such as CTB-60P12(obtainable from CalTech human BAC library B; GenBank accession numberAC002457), which contains the appropriate sequence.

Obtaining Human MDR-1 Coding Sequences

PCR Reaction 3: Production of an AvrII-ClaI fragment containing thehuman cDNA from within the 5′UTR to within the 3′UTR.

Human MDR-1 cDNA (GenBank Accession No. NM_(—)000927) is obtained byRT-PCR of human liver mRNA, using primers MDR3 and MDR6

Obtaining Human 3′ Flank

PCR Reaction 4: Production of a PmeI-XbaI Fragment Extending from theLast Exon to ˜1.7 kb Downstream of the Polyadenylation Signal.

PCR is performed with primers MDR7 and MDR8, using as a template, humangenomic DNA or more preferably, DNA from a BAC clone such as CTB-137N13(obtainable from CalTech human BAC library B; GenBank accession numberAC005068), which contains the appropriate sequence.

Assembling Vector

Step 1: The neomycin resistance gene is ligated into an appropriate site(e.g. SacII) in a pBluescript vector.Step 2: The PCR product from Reaction 1 is digested with SalI/ClaI andligated into a the SalI/ClaI sites of the vector from Step 1 (thus alsoinserting an AvrII site close to and upstream of the [ClaI] site).Step 3: The PCR product from Reaction 3 is digested with AvrII/ClaI andligated into the AvrII/ClaI sites of the vector from Step 2 (thus alsoinserting a PmeI site close to and upstream of the [ClaI] site).Step 4: The PCR product from Reaction 2 is digested with AvrII andligated into the AvrII site of the vector from Step 3.Step 5: The PCR product from Reaction 4 is digested with PmeI/XbaI andligated into the PmeI/XbaI sites of the vector from Step 4.

Generating Mice

The construct is linearised with NotI and transfected into ES cells forrandom integration (see Example 6). Alternatively the transgene can beremoved from the vector backbone by digestion with BssHII or SalI/XbaIprior to transfection. Clones are selected in G418 and analysed bySouthern blot to determine copy number. Suitable clones (preferably asingle gene copy) are implanted into blastocysts as described inprevious examples and the resultant chimeras bred to generate homozygousmice expressing human MDR-1. Such mice can be cross-bred with mdr 1a/1bdouble knock-out mice (Schinkel et al., 1997; Proc. Natl. Acad. Sci. USA94: 4028-4033), which are available from Taconic, Germantown, N.Y., USA.The resultant triple-transgenic mice will lack expression of both mouseMdr-1 genes but express the homologous human MDR-1 gene.

Example 6 Electroporation of ES Cells

Prior to electroporation day the following should be prepared:

-   (i) 12×10 cm plates and 4×6 cm plates containing a feeder layer of    mitotically inactivated neomycin resistant fibroblasts (or    hygromycin- or puromycin-resistant fibroblasts as appropriate.-   (ii) ES cells should be grown to approximately 80% confluency such    that at least 3×10⁷ cells are available on day of electroporation.-   1. Change media on the ES cells 2-4 hrs before cells will be    harvested. Usually 3×10 cm plates of ES cells will be available.    This will provide at least 4×10⁷ cells. This is more than adequate    since 2×10⁷ cells are required for one electroporation.-   2. Harvest cells as described previously using 0.25% trypsin/EDTA    and incubating for 5 min at 37° C.-   3. Collect cells into a 50 mL tube. Wash cells with EB media.-   4. Resuspend pellet in 10 mL of Electroporation Buffer (EB; 1× Hanks    solution (Gibco-BRL), 20 mM Hepes, 28 mM 2-beta mercaptoethanol, 1    mM NaOH).-   5. Determine viable cell density using trypan blue exclusion.-   6. Spin cells 1500 rpm for 5 min.-   7. Resuspend cells in EB such that cell density is approximately    3×10⁷ cells/mL.-   8. Label two, 0.4 cm electroporation cuvettes: (i) “+DNA” and add    2×10⁷ cells and 33 μg DNA in a final volume of 800 μL. (ii)-“DNA    control” and add 1.1×10⁶ cells in a final volume of 800 μL-   9. Allow to stand at room temperature for 10 min.-   10. Mix up and down gently with a sterile transfer pipette.-   11. Electroporate with gene pulsar with settings at 0.4 Kvolts, 25    μFD (time constant should be 0.4 or 0.5 sec).-   12. Allow to stand for 10 min at room temperature.-   13. Plate out cells from +DNA cuvette onto 12×10 cm plates, along    with the proportionate amount of cells onto the 2×6 cm control    plates.-   14. Plate out cells from −DNA cuvette onto 2×10 cm plates.-   15. Begin selection with geneticin (G418) alone or with the addition    of ganciclovir 24 hours later.-   16. For double selection usually use a concentration of 300 μg/mL    for G418 and 2 μM for ganciclovir.-   17. Change media daily.-   18. Pick surviving clones on day 10 or 11.

Example 7 Picking Colonies Materials:

-   -   Dissecting microscope, mouth pipette, multi-channel pipette, 96        well U-bottom plates    -   Day before picking prepare 15 or 30, 24 well plates with        mitotically inactivated neomycin resistant embryonic        fibroblasts. These cells are set up in ES cell growth media so        that they are ready to be used the following day.

Preparation:

-   -   Using a multichannel pipette add 30 μL of 0.25% trypsin-EDTA to        96 well U-bottom plates. Set up enough plates for the number of        cells to be picked i.e. 4×96 well plates when picking 360        colonies or 8×96 well plates when picking 720 colonies.    -   Add Hanks/Hepes buffer to 2 wells of a 6 well plate, this can be        used for washing the picking pipette in between colonies. Add        0.25% trypsin/EDTA to one well of the 6 well plate, a small        volume of this is collected into the picking pipette so that the        colony can be maintained in trypsin.

Procedure:

-   1. Wash 2 or 3 of the 10 cm plates that contain the colonies to be    picked with 5-10 mL of Hanks/Hepes buffer (H/H).-   2. Add 5 ml H/H and leave on plate.-   3. Wash pulled pasteur pipette a number of times with 70% ethanol.-   4. Wash repeatedly with H/H and then collect a small volume of    trypsin solution with picking pipette.-   5. Using the dissecting microscope, which is set up in a Laminar    flow cabinet, identify the colony that you want to pick.-   6. Gently cut around the feeder cell layer with the picking pipette.    Aspirate the ES cell colony into the pipette by mouth suction and    transfer the colony to one of wells of a 96 well plate containing    the trypsin solution.-   7. Collect 24 colonies (ie. 3 rows of a 96 well plate).-   8. Transfer the 96 well plate to the 37° C. incubator for 5 mins to    encourage the cells to disperse.-   9. Using a multichannel pipette disperse the colony by agitating    vigorously 2-3 times.-   10. Add 50 μL of media from the 24 well plate into the 96 well plate    and again disperse vigorously.-   11. Transfer all cells to 24 well plate, keeping each individual    colony separate.-   12. Continue until all 24 well plates have ES cells in them.-   13. Change media the following day.

Example 8 Construction of the pBluescript neo tk Vector

The pBluescript neo tk vector was constructed as follows. The neomycinresistance gene has a tk promoter and was excised from pMC1neo (GenBankAcc. No. U43612) with SalI and XhoI and inserted into the SmaI site ofpBluescript II KS. The thymidine kinase gene is derived from HerpesSimples Virus (HSV) and has been engineered for expression in ES cells(GenBank Acc. No. AF090451). It is flanked by a duplication of a mutantpolyoma virus enhancer. The thymidine kinase gene was excised from pIC19R/MC1-tk with XhoI and HindIII and cloned into the ApaI site of thepBluescript neo vector. Both the neo and tk genes and the T3 promoter ofpBluescript are transcribed in the same direction.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims.

TABLE 1 Common metabolic reactions involved in the biotransformation ofdrugs Biotransformation reaction Oxidative reactions Dealkylation (O- orN-linked) Deamination Desulphuration Hydroxylation (aliphatic oraromatic side chains) Hydroxylation (N-linked) Sulphoxidederivativisation Conjugation reactions Acetylation GlucuronidationGlycine conjugation Methylation (O-, N-, or S-linked) Sulphateconjugation Hydrolytic reactions Hydrolysis of esters or amidesReductive metabolism Azo groups Nitro groups Adapted from (Gilman etal., 1985, supra)

1. A method of predicting a behavior of a single drug or a plurality ofdrugs in a human, as part of a drug screening or evaluation process,comprising administering the single drug or the plurality of drugs to atransgenic mouse comprising in its genome a disruption of an endogenousmouse multidrug-resistance (MDR) gene and a stably integrated nucleotidesequence that encodes a human MDR polypeptide operably linked to apromoter, wherein the expression of the endogenous MDR gene is abrogatedor reduced, and wherein the human MDR polypeptide is other than theintended target of the single drug or plurality of drugs, and whereinexpression of the nucleotide sequence that encodes the human MDRpolypeptide provides a mouse that predicts behavior of a drug in ahuman, and conducting analytical tests to determine the behavior of thedrug in the transgenic mouse.
 2. The method of claim 1, wherein theresults of the analytical tests have a higher correlation to thebehavior of the drug in a human than the results obtained from a wildtype.
 3. The method of claim 1, wherein the human MDR polypeptide isMDR1 (P-glycoprotein).
 4. The method of claim 1, wherein the human MDRpolypeptide is a MRP.
 5. The method of claim 1, wherein the analyticaltest comprises assessing directly or indirectly, a concentration and/ordistribution of the drug in the transgenic mouse to which it has beenadministered.
 6. The method of claim 1, wherein the analytical testcomprises assessing directly or indirectly, an efficacy of the drug inthe transgenic mouse to which it has been administered.
 7. The method ofclaim 1, wherein the analytical test comprises assessing directly orindirectly, a toxicity of the drug in the transgenic mouse to which ithas been administered.
 8. The method of claim 1, wherein the analyticaltest comprises assessing directly or indirectly, a half-life of the drugin the transgenic mouse to which it has been administered.
 9. The methodof claim 1, wherein the analytical test comprises assessing directly orindirectly, a pharmacodynamics of the drug in the transgenic mouse towhich it has been administered.
 10. The method of claim 1, wherein theanalytical test comprises assessing directly or indirectly, apharmacokinetics of the drug in the transgenic mouse to which it hasbeen administered.
 11. The method of claim 1, wherein the analyticaltest is at least part of a drug-screening process.
 12. The method ofclaim 1, wherein the analytical test is at least part of a preclinicalassessment of a drug.
 13. The method of claim 1, wherein the analyticaltest is at least part of a drug-selection process.
 14. The method ofclaim 1, wherein the genome of the transgenic mouse comprisesreplacement of at least a portion of the endogenous mouse MDR gene withthe nucleotide sequence that encodes the human MDR polypeptide.
 15. Themethod of claim 1, wherein the genome of the transgenic mouse comprisesa disruption in the endogenous mouse MDR gene, wherein the disruptionresults in abrogated expression levels of the endogenous mouse MDR gene.16. The method of claim 1, wherein the genome of the transgenic mousecomprises a disruption in the endogenous mouse MDR gene, wherein themouse lacks the ability to produce a functional mouse MDR polypeptide.17. The method of claim 1, wherein the genome of the transgenic mousecomprises a disruption in the endogenous mouse MDR gene, wherein thedisruption comprises a deletion of at least a portion of the endogenousmouse MDR gene.
 18. The method of claim 1, wherein the promoter isendogenous to the mouse or in a human.
 19. The method of claim 1,wherein the promoter is an endogenous mouse MDR gene promoter.
 20. Themethod of claim 1, wherein the promoter is a human MDR gene promoter.21. The method of claim 1, wherein the nucleotide sequence is introducedby homologous recombination or introduction of a recombinase system intothe genome of the mouse.
 22. The method of claim 21, wherein therecombinase system is a Cre-loxP or FLP-FRT system.
 23. The method ofclaim 1, wherein the transgenic mouse is heterozygous for a transgenethat comprises the nucleotide sequence encoding the human MDRpolypeptide.
 24. The method of claim 1, wherein the transgenic mouse ishomozygous for a transgene that comprises the nucleotide sequenceencoding the human MDR polypeptide.